Fatty acids, coco, esters with 3,3'-oxybis[1,2-propanediol]

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:

2017-01-27 to 2017-08-04

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:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

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)

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.
One additional sample of the control was taken at test start and test end without any sample treatment.

Vehicle:

no

Details on test solutions:

Before test start the test media of each test concentration were prepared by dissolving test item in test water by stirring for about 48 hours. The highest test item concentration of 100 mg test item/L was prepared by mixing 101.4 mg test item into 1014 mL test water, for the test item concentration of 32 mg test item/L, 33.0 mg test item were mixed into 1031 mL test water, for the concentration of 10 mg test item/L, 10.5 mg were mixed into 1050 mL test water. The concentration of 3.2 mg test item/L was prepared by mixing 10.3 mg into 3219 mL test water and for 1.0 mg test item/L, 10.6 mg were mixed into 10600 mL test water. Then non-dissolved fractions of the test item were separated from the test media by membrane filtration (0.45 µm cellulose acetate filter). The pH of the highest concentration was adjusted from pH 6.4 to pH 7.8.
The test media were prepared just before introduction of the algae (= start of the test).

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.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

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

Test temperature:

21 to 22°C

pH:

7.2 to 8.0 at test start and 8.1 to 9.4 at test end

Nominal and measured concentrations:

Concentrations of nominal 100, 32, 10, 3.2 and 1.0 mg test item/L and a control were tested.
Each concentration was prepared by dissolving test substance in test water and filtration.
Corresponding to mean measured concentrations of 39.5, 9.56, 4.42 mg test item/L. The lower two concentrations of nominal 3.2 and 1.0 mg test item/L were below LOQ. In the control, test water was used without addition of the test item.

Details on test conditions:

Water temperature was: 21.4 to 22.1 °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: 7.2 to 8.0 at test start and 8.1 to 9.4 at test end
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: 6182 lux (range: 5970 - 6740 lux)

Reference substance (positive control):

yes

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

17.57 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

3.14 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

4.42 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

9.56 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.65 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

5.53 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

7.19 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

4.42 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

9.56 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

The 72-hour EyC50 was calculated to be 7.19 mg test item/L and the ErC50 17.57 mg test item/L. The 72-hour EyC10 was calculated to be 4.65 mg test item/L and the ErC10 n.d. mg test item/L. The 72-hour NOEyC was determined to be 4.42 mg test item/L and the associated 72-hour LOEyC of 9.56 mg test item/L. The 72-hour NOErC was determined to be 4.42 mg test item/L and the associated 72-hour LOErC is 9.56 mg test item/L.

Results with reference substance (positive control):

Parameter 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 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 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 Weibull analysis using linear max. 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 two lowest concentrations below LOQ were not included in the statistical analysis. For the highest concentration above water solubility the measured concentration was used for statistical analyses.
The software used to perform the statistical analysis was ToxRat Professional, Version 2.10.05, ToxRat Solutions GmbH.

Validity criteria fulfilled:

yes

Conclusions:

72-EC50 (Pseudokirchneriella subcapitata, growth rate) = 17.57 mg test item/L; 72h-NOErC (Pseudokirchneriella subcapitata, growth rate) = 4.42 mg test item/L; 72h-LOErC (Pseudokirchneriella subcapitata, growth rate) = 9.56 mg test item/L (mean measured)

Executive summary:

The purpose of this test was to determine the inhibitory effect of the test item Hostastat FE 20 LIQ on the growth of the freshwater green algal species 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: Hostastat FE 20 LIQ; Batch No.: ESD0021912; purity: 100 % (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: Concentrations of nominal 100, 32, 10, 3.2 and 1.0 mg test item/L and a control were tested.

Each concentration was prepared by dissolving test substance in test water and filtration.

Corresponding to mean measured concentrations of 39.5, 9.56, 4.42 mg test item/L. The lower two concentrations of 3.2 and 1.0 mg test item/L were below LOQ.

Test Conditions: Water temperature: 21.4 to 22.1 °C; pH values at test start 7.2 to 8.0, at the end of the test 8.1 to 9.4; continuous illumination; mean light intensity: 6182 lux (5970 to 6740 lux).

Results

The influence of Hostastat FE 20 LIQ 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 7.19 mg test item/L and the 72-hour ErC50 value was calculated to be 17.57 mg test item/L. The 72-hour NOEyC was determined to be 4.42 mg test item/L and the associated 72-hour LOEyC was 9.56 mg test item/L. The 72-hour NOErC was determined to be 4.42 mg test item/L and the associated 72-hour LOErC was 9.56 mg test item/L. The quantification of the test item Hostastat FE 20 LIQ in the test samples was performed by analysing the Total Organic Carbon content (TOC).

This study was classified acceptable and satisfied the guideline requirements for growth inhibition algae studies.

Description of key information

72-EC50 (Pseudokirchneriella subcapitata, growth rate) = 17.57 mg test item/L; 72h-NOErC (Pseudokirchneriella subcapitata, growth rate) = 4.42 mg test item/L; 72h-LOErC (Pseudokirchneriella subcapitata, growth rate) = 9.56 mg test item/L (mean measured, OECD 201, 2017)

Key value for chemical safety assessment

EC50 for freshwater algae:

17.57 mg/L

EC10 or NOEC for freshwater algae:

4.42 mg/L

Additional information

In a reliable key study, toxicity of the submission substance ongrowth of the freshwater green algal was investigated (RL1). Growing cultures of unicellular green algal were exposed to five test concentrations (nominal 100, 32, 10, 3.2 and 1.0 mg test item/L) under static test conditions during 72 hours. A control was run in parallel. The test concentrations were analytically verified by analysing the Total Organic Carbon content (TOC). The corresponding mean measured concentrations were 39.5, 9.56, 4.42 mg test item/L, and the lowest two concentrations of 3.2 and 1.0 mg test item/L were below LOQ. Reported results refer to mean measured concentrations.

Adverse effects on growth rate and yield were observed at the two highest test concentrations, i.e. 39.5 and 9.56 mg/L test item. Effects occurred in a dose-response relationship. Derived effect concentration on yield (y) and growth rate (r) were:

72h-EyC50 = 7.19 mg/L

72h-LOEyC = 9.56 mg/L

72h-NOEyC = 4.42 mg/L

72-ErC50= 17.57 mg/L

72h-LOErC = 9.56 mg/L

72h-NOErC = 4.42 mg/L

 

This study was classified acceptable and satisfied the guideline requirements for toxicity to microorganisms studies. In conclusion, the test was considered reliable and adequate for the environmental hazard assessment for aquatic organisms.