2-ethyl-3-hydroxy-4-pyrone

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:

04 November 2016 - 10 March 2017

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:

2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control, 1.0, 3.2, 10, 32, 100 mg/L
- Sampling method: The concentration and stability of the test item in the test preparations were verified by chemical analysis. Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. Duplicate samples were taken at 0 and 72 hours and stored for further analysis if necessary. Two additional samples of each test concentration were prepared at the start of the test and incubated alongside the provide samples for analysis at 24 and 48 hours should the need arise.
- Sample storage conditions before analysis: The samples were stored frozen prior to analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
A nominal amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1 liter to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 32, 10, 3.2 and 1.0 mg/L. An aliquot (500 mL) of each of the stock solutions was separately inoculated with algal suspension (4.6 mL) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L. The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source: SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.

ACCLIMATION
- Pre-culture: Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10^4 – 10^5 cells/mL.
- Method of cultivation: The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 2 °C.
- Culturing media and conditions (same as test or not): The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

Temperature was maintained at 24 ± 1 ºC throughout the test.

pH:

t = 0h: 7.2 - 7.7
t = 72h: 6.6 - 8.1

Nominal and measured concentrations:

Final test:
Nominal concentrations: 0, 1.0, 3.2, 10, 32 and 100 mg/L.
Analytical concentrations:
0h: 72h: Geometric Mean Measured Test Concentrations: 0, 0.21, 0.77, 2.9, 14, 66 mg/L

In cases where the measured concentration was less than the LOQ of the analytical method a value of half the LOQ (i.e. 0.045 mg/L) was used to enable calculation of the geometric mean measured concentration.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks.
- Type (delete if not applicable): closed, plugged with polyurethane foam bungs.
- Fill volume: 100 mL
- Aeration: flasks were constantly shaken at approximately 150 rpm for 72 hours.
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Initial cells density: Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 5.46 x 10^5 cells per mL. Inoculation of 500 mL of test medium with 4.6 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10^3 cells per mL and had no significant dilution effect on the final test concentration.
- Control: The control group was maintained under identical conditions but not exposed to the test item.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The culture medium was prepared using reverse osmosis purified deionized water.
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The appearance of the test media was recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: the pH adjusted to 7.5 with 0.1N NaOH or HCl.
- Photoperiod: continuous illumination.
- Light intensity and quality: intensity approximately 7000 lux provided by warm white lighting (380 – 730 nm).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples were taken at 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00 x 10^3 cells/mL) was taken as the starting cell density.
- Chlorophyll measurement: No
- Other: To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

TEST CONCENTRATIONS
- Range finding study: Yes
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes, the results showed no effect on growth rate at the test concentrations of 0.10 and 1.0 mg/L. However, growth was observed to be reduced at 10 and 100 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

7.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL 5.9 - 8.9 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.77 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Inhibition of growth rate
ErC10 (0 - 72 h) : 1.8 mg/L
ErC20 (0 - 72 h) : 3.0 mg/L
ErC50 (0 - 72 h) : 7.2 mg/L; 95% confidence limits 5.9 – 8.9 mg/L
NOEC (0 - 72 h): 0.77 mg/L

Inhibition of yield:
EyC10 (0 - 72 h) : 2.3 mg/L
EyC20 (0 - 72 h) : 2.4 mg/L
EyC50 (0 - 72 h) : 2.8 mg/L; 95% confidence limits 2.6 – 2.9 mg/L
NOEC (0 - 72 h): 0.77 mg/L

Validation criteria:
• The cell concentration of the control cultures increased by a factor of 115 (> 16) after 72 hours.
• The mean coefficient of variation for section by section specific growth rate for the control cultures was 14% (< 35%)
• The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 3% (< 7%)

All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 1.0, 3.2, 10 and 32 mg/L, however no intact cells were observed to be present in the test cultures at 100 mg/L.
At the start of the test all control and test cultures were observed to be clear coloress solutions. After the 72-Hour test period all control, 1.0 and 3.2 mg/L test cultures were observed to be green dispersions. The 10 mg/L test cultures were observed to be pale green dispersions whilst the 32 and 100 mg/L test cultures were observed to be clear colorless solution

Results with reference substance (positive control):

ErC50 (0 – 72 h) : 1.4 mg/L; 95% confidence limits 1.2 – 1.5 mg/L.
EyC50 (0 – 72 h) : 0.60 mg/L; 95% confidence limits 0.52 – 0.69 mg/L.

The results were within the normal ranges for this reference item.

Reported statistics and error estimates:

Statistical Analysis:
One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the growth rate and yield data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups.

Table 1. Inhibition of Growth Rate and Yield in the Definitive Test

Nominal concentration (mg/L)	Geometric mean concentration (mg/L)	Inhibition % growth rate	Inhibition % yield
Control	0	-	-
1	0.21	(3)	(15)
3.2	0.77	(3)	(17)
10	2.9	21	62
32	14	73	98
100	66	97	100

(x) indicates increase of growth/yield

Validity criteria fulfilled:

yes

Remarks:

see details on results

Conclusions:

72 h exposure of Pseudokirchneriella subcapitata to the test item gave an ErC10 and ErC50 of 1.8 and 7.2 mg/L (based on geometric mean measured concentrations), respectively.

Executive summary:

A fresh water alga and cyanobacteria growth inhibition test was performed in accordance with OECD guideline 201 under GLP. Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to the test item at nominal concentrations of 0, 1.0, 3.2, 10, 32 and 100 mg/L for 72 hours at 24 °C. The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 hours and from the pooled replicates at 72 hours. The geometric mean measured test concentrations were determined to be 0.21, 0.77, 2.9, 14 and 66 mg/L. Under the conditions of the test, the test item gave an ErC10 and ErC50 of 1.8 and 7.2 mg/L (based on geometric mean measured concentrations), respectively. The NOEC for biomass and growth rate was 0.77 mg/L.

Description of key information

A fresh water alga and cyanobacteria growth inhibition test was performed in accordance with OECD guideline 201 under GLP. Following a preliminary range-finding test,Pseudokirchneriella subcapitatawas exposed to the test item at nominal concentrations of 0, 1.0, 3.2, 10, 32 and 100 mg/L for 72 hours at 24 °C. The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 hours and from the pooled replicates at 72 hours. The geometric mean measured test concentrations were determined to be 0.21, 0.77, 2.9, 14 and 66 mg/L. Under the conditions of the test, the test item gave an ErC10 and ErC50 of 1.8 and 7.2 mg/L (based on geometric mean measured concentrations), respectively. The NOEC for biomass and growth rate was 0.77 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

7.2 mg/L

EC10 or NOEC for freshwater algae:

1.8 mg/L