Fatty acids, C18-unsatd., compds. with triethanolamine

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-02-07 to 2017-08-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Guideline Study; EU method; GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OECD No 23, Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C18 unsatd., reaction products with triethanolamine

EC Number:

938-513-5

Cas Number:

1187872-27-0

IUPAC Name:

Fatty acids, C18 unsatd., reaction products with triethanolamine

Test material form:

liquid: viscous

Specific details on test material used for the study:

Name: Emulsogen TO
CAS No.: 10277-04-0
Certificate of Analysis Date: September 01, 2016
Expiry Date: September 01, 2018
Aggregate State at Room Temperature: Liquid
Colour: brown
Storage Conditions at Test Facility:
At 20 ± 5 °C, in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Duplicate samples from the freshly prepared test media (containing algae) of all test concentrations and from the control were taken at the start of the test.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test media of all test concentrations and the control (containing algae) were taken at the end of the test (after the 72 hours test period) by pouring together the contents of the test beakers of each treatment and the additional vessels for analytical sampling.

Test solutions

Vehicle:

no

Details on test solutions:

Due to the low limit of water solubility of the test item Water accommodated Fractions (WAF) were prepared for each concentration separately. The nominal concentrations were prepared by mixing 100.0, 32.0, 10.0, 9.6 and 10.0 mg test item/L into 1000, 1000, 1000, 3000 and 10000 mL test water, respectively. After carefully stirring for 48 hours in the dark at room temperature a settling period of 1 hour followed to allow phase separation. The aqueous phase, i.e. the water accommodated fraction, was drawn off carefully and used as the test medium of the corresponding nominal test concentration.
The test media were prepared just before introduction of the algae (= start of the test).

Test organisms

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata (KORSHIKOV), Strain No. 61.81 SAG, formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV)
Origin: The algae were supplied by the „Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen", 37073 Göttingen, Germany.
Breeding Conditions: The algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L) as CaCO3

Test temperature:

22 to 23°C

pH:

7.9 to 8.1 at test start and 7.8 to 10.3 at test end

Nominal and measured concentrations:

Test Concentrations: Due to the low limit of water solubility of the test item Water accommodated Fractions (WAF) with nominal loading rates of 100, 32, 10, 3.2 and 1.0 mg/L and a control were tested.
Control: In the control, test water was used without addition of the test item.

Details on test conditions:

Water Temperature:
The temperature was measured daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks.
Water temperature was: 21.5 to 22.8 °C
pH-Values: The pH was measured in all test item concentrations and the control at the start and the end of the test.
pH was: 7.9 to 8.1 at test start and 7.8 to 10.3 at test end
The pH in the control increased by more than 1.5 units in the control medium. However, this does not invalidate the tests since the validity criterion was met.
Light Regime: Continuous illumination
Light Intensity: The light intensity was measured once during the test at 6 positions distributed over the experimental area at the surface of the test media.
Mean light intensity: 6838 lux (range: 6430 to 7200 lux)

Reference substance (positive control):

yes

Remarks:

reference item potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

20.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

9.11 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 4

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

51.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

17.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 7

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 8

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 9

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

12.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 10

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

25.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The 72-hour EyC50 was calculated to be 20.9 mg test item/L and the ErC50 51.5 mg test item/L. The 72-hour EyC10 was calculated to be 9.11 mg test item/L and the ErC10 17.9 mg test item/L. The 72-hour NOEyC was determined to be 10.0 mg test item/L and the associated 72-hour LOEyC of 32 mg test item/L. The 72-hour NOErC was determined to be 10.0 mg test item/L and the associated 72-hour LOErC is 32 mg test item/L. All values refer to nominal concentrations.

Results with reference substance (positive control):

Parameter
(0-72 h) Yield
[mg test item/L]
72-hour EC50 0.549
95 % conf. interval 0.490 – 0.615

72-hour EC20 0.351
95 % conf. interval 0.301 – 0.412

72-hour EC10 0.278
95 % conf. interval 0.226 – 0.341

72-hour NOEC 0.21
72-hour LOEC 0.45

Parameter
(0-72 h) Growth rate
[mg test item/L]
72-hour EC50 1.12
95 % conf. interval 1.06 – 1.17

72-hour EC20 0.694
95 % conf. interval 0.646 – 0.747

72-hour EC10 0.541
95 % conf. interval 0.491 – 0.596

72-hour NOEC 0.21
72-hour LOEC 0.45

Parameter
(0-72 h) Biomass
[mg test item/L]
72-hour EC50 0.567
95 % conf. interval 0.503 – 0.638

72-hour EC20 0.347
95 % conf. interval 0.295 – 0.411

72-hour EC10 0.269
95 % conf. interval 0.217 – 0.334

72-hour NOEC 0.21
72-hour LOEC 0.45

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 Probit analysis using linear maximum likelihood regression.
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.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The influence of Emulsogen TO on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test. The 72-hour ErC50 value was calculated to be 51.5 mg test item/L. The 72-hour NOErC was determined to be 10 mg test item/L and the associated 72-hour LOErC was 32 mg test item/L.
The quantification of the test item Emulsogen TO in the test samples was performed using total organic carbon measurement (TOC).

Executive summary:

Title: Emulsogen TO: Toxicity to Pseudokirchneriella subcapitata in an Algal Growth Inhibition Test

Purpose: The purpose of this test was to determine the inhibitory effect of the test item Emulsogen TO on the growth of the freshwater green algae Pseudokirchneriella subcapitata.

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 determine the concentrations of the test item in the test medium.

Guidelines/Recommendations:

OECD Guideline for Testing of Chemicals, Section 2, No. 201: "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", adopted March 23, 2006, corrected July 28, 2011

Commission Regulation (EC) No 761/2009, Annex, Part C, C.3.: "Freshwater Algae and Cyanobacteria, Growth Inhibition Test", Official Journal of the European Union (EN), dated August 24, 2009

OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures", December 15, 2000

SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414

 

Material and Methods

Test Item: Emulsogen TO; Batch No.: DEB2 207167; purity: 99.5 % (w/w), according to certificate of analysis.

Test Species: Pseudokirchneriella subcapitata, Strain No. 61.81 SAG formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV).

Cultivated in the laboratories of ibacon; original source: "Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissen-schaften, Universität Göttingen", 37073 Göttingen, Germany.

Test Design: 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.

Endpoints: Yield and growth rate of the algae

Test Concentrations: Due to the low limit of water solubility of the test item Water accommodated Fractions (WAF) with nominal loading rates of 100, 32, 10, 3.2 and 1.0 mg/L and a control were tested.

Test Conditions: Water temperature: 21.5 to 22.8 °C; pH values at test start 7.9 to 8.1, at the end of the test 7.8 to 10.3; continuous illumination; mean light intensity: 6838 lux (6430 to 7200 lux).

Results:               

The influence of Emulsogen TO on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test. The 72-hour EyC50 was calculated to be 20.9 mg test item/L and the 72-hour ErC50 value was calculated to be 51.5 mg test item/L. The 72-hour NOEyC was determined to be 10 mg test item/L and the associated 72-hour LOEyC was 32 mg test item/L. The 72-hour NOErC was determined to be 10 mg test item/L and the associated 72-hour LOErC was 32 mg test item/L.

The quantification of the test item Emulsogen TO in the test samples was performed using total organic carbon measurement (TOC). The concentrations of the test item were determined in the duplicate test media samples from all test concentrations and the duplicate control samples from all sampling times. However, the measured TOC values in the three lowest test concentrations of nominal loading rate 10, 3.2 and 1.0 mg test item/L were below the limit of quantification. The organic carbon analysis showed that the organic carbon concentrations did not decrease over the course of the test. The data was not used to calculate test item recoveries. The report is therefore based on nominal test concentrations.

This study is classified acceptable and satisfies the guideline requirements for growth inhibition algae studies.