2-ethylhexyl 3-mercaptopropionate

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:

July 27, 2018 - December 19, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

Due to the low limit of water solubility of the test item a supersaturated stock solution of nominal 1.0 mg test item/L and dilutions with nominal concentrations of 0.40, 0.16, 0.064 and 0.026 mg/L and a control will be tested.
This stock solution and its dilutions correspond to the following geometric mean measured and initial concentrations:
0.0393, 0.0260, 0.0162, 0.0100 and 0.0100 mg test item/L and 0.496, 0.198, 0.0794, 0.0317 and 0.0127 mg test item/L.
A first main test was performed with the following concentrations: A supersaturated stock solution of nominal 1.0 mg test item/L and dilutions with nominal concentrations of 0.40, 0.16, 0.064 and 0.026 mg/L.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: Pseudokirchneriella subcapitata (KORSHIKOV), No. 61.81 SAG
- Source (laboratory, culture collection): „Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen", 37073 Göttingen, Germany
- Age of inoculum (at test initiation): These cells were taken from an exponentially growing pre-culture, which was set up 3 days prior to the test start under the same conditions as in the test.
- Method of cultivation: The algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21.3 to 23.2 °C

pH:

7.9 to 8.1 at test start and 9.0 to 10.0 at test end

Nominal and measured concentrations:

nominal concentrations: 0.40, 0.16, 0.064 and 0.026 mg/L
geometric mean measured concentrations: 0.0393, 0.0260, 0.0162, 0.0100 and 0.0100 mg test item/L
initial measured concentrations: 0.496, 0.198, 0.0794, 0.0317 and 0.0127 mg test item/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks of 50 mL volume with approximately 50 mL of test medium
- Initial cells density: approx. 5000 algal cells per mL
- Control end cells density:
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
The flasks were covered with air permeable glass dishes and incubated in a water bath. The flasks were placed in a random order and were repositioned each day to minimize differences in test conditions.
Additionally, one replicate of each test concentration and of the control was prepared without algae to provide a "blank" for the spectrophotometric measurements. The additional replicates were incubated under the same conditions as described above. The blank values were subtracted from the absorption measured in the samples containing algae in order to eliminate absorption caused by the test item.

GROWTH MEDIUM
- Standard medium used: yes


OTHER TEST CONDITIONS
- Photoperiod: Continuous illumination
- Light intensity and quality: 5313 lux (range: 4740 to 6090 lux)


EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: spectrophotometer, after 24, 48 and 72 hours

Reference substance (positive control):

yes

Remarks:

For the evaluation of the quality of the algae and the experimental conditions the reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 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.:

0.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.496 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.039 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.496 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.039 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: no

Results with reference substance (positive control):

- Results with reference substance valid?
- EC50: 72-hour EyC50 = 0.442 mg/L; ErC50 = 1.11 mg/L; EbC50 = 0.487 mg/L

Reported statistics and error estimates:

Based on the calculated cell densities, the 72 hours ErC50 and the 72 hours EyC50 (see Definitions), the corresponding EC20 and EC10 values and where possible their 95 %-confidence limits were calculated by logit analysis.
For the determination of the 72 hours LOEC and the 72 hours NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Williams t-test.
The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

Validity criteria:

Cell Density Increase in Control Cultures: 284.8-fold increase within 72 hours and thus, the validity criterion was met.
Coefficient of Variation of Sectional (Daily) Growth Rates in Control Cultures: 31.3 % and thus, the validity criterion was met.
Coefficient of Variation of Average Growth between Control Replicates: 1.2 % and thus, the validity criterion was met.

Microscopic Examination:
The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing up to a nominal test concentration of 1.0 mg test item/L and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to this test concentration, the highest concentration tested.

Validity criteria fulfilled:

yes

Conclusions:

72 h ErC50 > 1 mg/L (nominal), 72 h NOErC = 0.4 mg/L (nominal)

Executive summary:

The purpose of this test was to determine the inhibitory effect of the test item EHMP on the growth of the freshwater green algae Pseudokirchneriella subcapitata. The study was conducted according to OECD TG 201.
For this purpose, exponentially growing cultures of this unicellular green algal species were exposed to various concentrations of the test item under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations.
The test method of application and the test system are recommended by the test guidelines and Pseudokirchneriella subcapitata is one of the recommended test species.
The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium.

This study encompassed 6 treatment groups (5 dose rates of the test item and a control) with three replicates per test concentration and six replicates for the control. At test start 50 mL of the test concentrations were inoculated with approx. 5000 algal cells per mL test medium and defined volumes of the algal suspensions were sampled after 24, 48 and 72 hours for determination of cell densities by spectrophotometrical measurement.

Due to the low limit of water solubility of the test item a supersaturated stock solution of nominal 1.0 mg test item/L and dilutions with nominal concentrations of 0.40, 0.16, 0.064 and 0.026 mg/L and a control will be tested.
This stock solution and its dilutions correspond to the following geometric mean measured and initial concentrations:
0.0393, 0.0260, 0.0162, 0.0100 and 0.0100 mg test item/L and 0.496, 0.198, 0.0794, 0.0317 and 0.0127 mg test item/L.

The quantification of the test item EHMP in the test samples was performed using liquid chromatography with UV detection.
At the start of the test 50 % of the filtrate of the nominal test concentration of 100 mg test item/L were found. No signals could be determined in the samples of test end.

The 72-hour EyC50 was calculated to be > 0.0393 mg test item/L (> 0.496 mg test item/L), and the 72-hour ErC50 value was calculated to be > 0.0393 mg test item/L (> 0.496 mg test item/L). The 72-hour NOEyC was determined to be 0.0260 mg test item/L (0.198 mg test item/L) and the associated 72-hour LOEyC was 0.0393 mg test item/L (0.496 mg test item/L). The 72-hour NOErC was determined to be 0.0260 mg test item/L (0.198 mg test item/L) and the associated 72-hour LOErC was 0.0393 mg test item/L (0.496 mg test item/L). Statistically significant effects on the freshwater algae could be observed in the highest test concentration of 0.0393 mg test item/L (0.496 mg test item/L) for both endpoints (yield and growth rate). Values refer to geometric mean measured test concentrations, values in parentheses refer to initial test concentrations.

However, since the measured values don't represent the real environmental conditions because the test substance is rapidly oxidized by the oxygen content in the aqueous phase. As the transformation products are less toxic than the parent substances (no free –SH groups) regulatory endpoints calculated on the basis of nominal concentrations represent a realistic worst case approach. 

The nominal 72 h ErC50 was >  1 mg/L, the 72 h ErC10 was >  1 mg/L, the 72 h NOErC = 0.4 mg/L.

 

 

Description of key information

72 h ErC50 > 1 mg/L, the 72 h ErC10 was > 1 mg/L, the 72 h NOErC = 0.4 mg/L, based on nominal concentrations

Key value for chemical safety assessment

EC50 for freshwater algae:

1 mg/L

EC10 or NOEC for freshwater algae:

1 mg/L

Additional information

The purpose of this test was to determine the inhibitory effect of the test item EHMP on the growth of the freshwater green algae Pseudokirchneriella subcapitata. The study was conducted according to OECD TG 201.
For this purpose, exponentially growing cultures of this unicellular green algal species were exposed to various concentrations of the test item under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations.
The test method of application and the test system are recommended by the test guidelines and Pseudokirchneriella subcapitata is one of the recommended test species.
The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium.

This study encompassed 6 treatment groups (5 dose rates of the test item and a control) with three replicates per test concentration and six replicates for the control. At test start 50 mL of the test concentrations were inoculated with approx. 5000 algal cells per mL test medium and defined volumes of the algal suspensions were sampled after 24, 48 and 72 hours for determination of cell densities by spectrophotometrical measurement.

Due to the low limit of water solubility of the test item a supersaturated stock solution of nominal 1.0 mg test item/L and dilutions with nominal concentrations of 0.40, 0.16, 0.064 and 0.026 mg/L and a control will be tested.
This stock solution and its dilutions correspond to the following geometric mean measured and initial concentrations:
0.0393, 0.0260, 0.0162, 0.0100 and 0.0100 mg test item/L and 0.496, 0.198, 0.0794, 0.0317 and 0.0127 mg test item/L.

The quantification of the test item EHMP in the test samples was performed using liquid chromatography with UV detection.
At the start of the test 50 % of the filtrate of the nominal test concentration of 100 mg test item/L were found. No signals could be determined in the samples of test end.

The 72-hour EyC50 was calculated to be > 0.0393 mg test item/L (> 0.496 mg test item/L), and the 72-hour ErC50 value was calculated to be > 0.0393 mg test item/L (> 0.496 mg test item/L). The 72-hour NOEyC was determined to be 0.0260 mg test item/L (0.198 mg test item/L) and the associated 72-hour LOEyC was 0.0393 mg test item/L (0.496 mg test item/L). The 72-hour NOErC was determined to be 0.0260 mg test item/L (0.198 mg test item/L) and the associated 72-hour LOErC was 0.0393 mg test item/L (0.496 mg test item/L). Statistically significant effects on the freshwater algae could be observed in the highest test concentration of 0.0393 mg test item/L (0.496 mg test item/L) for both endpoints (yield and growth rate). Values refer to geometric mean measured test concentrations, values in parentheses refer to initial test concentrations.

However, since the measured values don't represent the real environmental conditions because the test substance is rapidly oxidized by the oxygen content in the aqueous phase. As the transformation products are less toxic than the parent substances (no free –SH groups) regulatory endpoints calculated on the basis of nominal concentrations represent a realistic worst case approach. 

The nominal 72 h ErC50 was >  1 mg/L, the 72 h ErC10 was >  1 mg/L, the 72 h NOErC = 0.4 mg/L.