Hexan-4-olide

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

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)

GLP compliance:

yes

Analytical monitoring:

yes

Vehicle:

yes

Remarks:

growth medium

Details on test solutions:

Growth medium
To prepare the growth medium, 4 vials (A, B, C, D) of different concentrated saIts "ISO medium” (purchased from ECOTOX LDS), were used; each vial contained a concentrated salt solution enough to prepare 1 I of growth medium. The composition of each vial is given below.
Batch used: SC040417
Expiration date: 04/04/2019
NRI: 173VIT/2018 and 215VIT/2018
A. Vial A: macro-nutrients:
NH4CI 1.5 /l - dissolved in 1 I - final concentration 15 mg/I
MgCI, 6H20 1.2 g/1 - dissolved in 1 l - final concentration 12 mg/I
CaCl2 2H20 1.8 g/1 - dissolved in 1 I - final concentration 18 mg/I
MgS04 7H20 1.5 g/1 - dissolved in 1 I - final concentration 15 mg/I
KH2PO4 0.16 g/1 - dissolved in 1 I - final concentration 1.6 mg/
B. Vial B: Iron:
FeCl3 6H2O 64 mg/I - dissolved in 1 I - final concentration 64 ug/l
Na2EDTA 2H20 100 mg/I…dissolved in 1 l - final concentration 100 µg/l
C. Vial C: trace elements:
H3B03 185 mg/I - dissolved in 1 l - final concentration 185 µg/1
MnCl2 4H2O 415 mg/I - dissolved in 1 I - final concentration 415 µg/1
ZnCl2 3 mg/I - dissolved in 1 I - final concentration 3ug/l
CoCl2 6H2O 1.5 mg/I - dissolved in 1 I - final concentration 1.5ug/1
CuCl2 2H2O 0.01 mg/I - dissolved in 1 I - final concentration 0.01ug/1
Na2MoO4 2H2O 7 mg/I - dissolved in 1 I - final concentration 7 µg/1
D Vial D: bicarbonate
NaHCO3 50 g/l - dissolved in 1 I - final concentration 50 mg/I The growth medium was prepared as follows:
A volumetric flask (1 I capacity) was filled with approximately 800 ml of distilled water;
10 ml of solution A and 1 ml of solutions B, C and D were added to the flask;
The flask was filled up to 1 I with distilled water and mixed.
The growth medium was transferred in dark glass containers and stored in the refrigerator (5 °C ± 3 °C), in the dark, for no longer than one month.
The pH of the growth medium was in the acceptable range of 8.1±0.2. Before using the growth medium for the de­immobilization of algae or for the preparation of the test solutions, it was gradually brought to room temperature, and it was aerated for 30 minutes.
At the beginning of each test, pH of the growth medium was measured.
The compliance of pH value was reported in the raw data of the Study.

K2Cr2O7
Prior to use, the batch of algae was subjected to a full test using potassium dichromate, in order to verify the sensitivity of the batch as indicated in the certificate. The results were reported in the AR 2017/025/B.

Reagents for TOC analysis
Sucrose
1,4-Benzoquinone
Nitric acid concentrate 70% (for washing volumetric ftasks needed for the solutions employed in the analaysis)
Sucrose solutions were freshly prepared prior to begin with the analysis at each time point
At first the sucrose was heated in a oven for at least 3 hours at 105 °C and afterwards dried for at least 1 hour A 5000 ppm working solution (RSO) was prepared by solving the sucrose in MilliQ H2O.
FR 17,612119,0006
From the 只SO solution, 1ml was transferred in a new 100 ml volumetric flask and brought to volume with MillliQ H20 in order to prepared the RS0-50 ppm solution.
The other dilutions were prepared as follows:
RS1 (0.2 ppm) = 0.4 ml of RS0-50 ppm were transferred into a 100 ml volumetric flask and brought to volume with milliQ H20;
RS2 (1 ppm) = 2 ml of RS0-50 ppm were transferred into a 100 ml volumetric flask and brought to volume with milliQ H20;
RS3 (2.5 ppm) = 5 ml of RS0-50 ppm were transferred into a 100 ml volumetric flask and brought to volume with milliQ H20;
RS4 (5 ppm) = 10 ml of RS0-50 ppm were transferred into a 100 ml volumetric flask and brought to volume with milliQ H20;
RS5 (10 ppm) = 20 ml of RS0-50 ppm were transferred into a 100 ml volumetric flask and brought to volume with milliQ H20;
RS6 (20 ppm) = 40 ml of RS0-50 ppm were transferred into a 100 ml volumetric flask and brought to volume with milliQ H20.
A 5000 ppm 1,4-Benzoquinone stock solution (BQ0 - 5000 ppm) was prepared in MilliQ H20 and the solution was then sonicated for 10 minutes.
Afterwards BQ1 (50 ppm) solution was prepared by transferring 1 ml of BQ0 into a new 100 ml volumetric flask and bringing to volume with MilliQ H20.
Finally BQ2 (0.5 ppm) solution was prepared by transferring 1 ml of BQ1 into a new 100 ml volumetric flask and flask and bringing to volume with MilliQ H20.

Test organisms (species):

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

Details on test organisms:

As test system Selenastrum capricornutum (formerly known as Pseudokirchneriel/a subcapitata) ATCC 22662, CCAP 278/4, 61.81 SAG, was employed in the assay, as described by the guideline OECD 201:2006 corr. 2011. Algal cultures were supplied from the qualified provider ECOTOX LDS, in a quiescent form (algal beads), and they can be activated when needed. The use of a quiescent form of the algal cultures eliminates the need of maintaining continuous cultures.
The vials with the algal beads were stored in the refrigerator at 5 "C+-2 'C; they were provided with a quality control certificate which reported the test species, the batch number, the expiration date, the storage and activation conditions and the EC50 - 72 h value for K2Cr2O7. As internal quality control EC50 value was determined for the batch of algae used, and reported in the AR 2017/025/B. For this test, the following algal beads were used:
Batch: SC141217 (Tl-0015464 and Tl-0015465)
Expiration date: 14/05/2018

Test type:

not specified

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

21 °C ± 2 °C

pH:

7.44-7.91

Reference substance (positive control):

yes

Remarks:

K2Cr2O7

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 13.06 mg/L

Basis for effect:

biomass

Details on results:

The EC50 value was calculated with mod 720/GxP data-sheet and resulted to be 13.06 mg/I (effective titer). In this case, EC50 value was considered valid since obtained results of inhibition growth were in the range between 10 % and 90 %.
The graph of the fitted model used for the calculation of the EC50 is attached to the raw data.

Results with reference substance (positive control):

Prior to routine use of thebatch of Selenastrum capricornutum. the sensitivity toward K2Cr2O7 was verified by carrying out a test with 5 concentrations of this chemical; the concentrations used in this test were: 1.8 mg/I - 1 mg/I - 0.56 mg/I - 0.32 mg/I - 0.18 mg/I. Results of the tests were reported in AR 2017102518.

Validity criteria fulfilled:

yes

Conclusions:

According to OECD 201 :2006 corr. 2011, under the test conditions applied, the EC50 of the test substance is calculated as 13.06 mg/l (effective titer).

Key value for chemical safety assessment

EC50 for freshwater algae:

13.06 mg/L