(Z)-tetrahydro-6-(2-pentenyl)-2H-pyran-2-one

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:

23 April 2019 - 31 May 2019

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)

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

Remarks:

(incl. certificate)

Details on sampling:

- Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a haemocytometer and light microscope.
- All samples were taken for immediate analysis. Duplicate sets of samples were taken at 0 and 72 hours and stored frozen for further analysis if required.

Vehicle:

no

Details on test solutions:

PREPARATION OF TEST SOLUTION
A nominal amount of test item (200 mg) was dissolved in culture medium and the volume adjusted to 2 liters to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 1.0, 3.2, 10 and 32 mg/L. An aliquot (900 mL) of each of the stock solutions was separately inoculated with algal suspension (5.6 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.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 48 and 72 hours using high performance liquid chromatography with mass spectrometry.

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: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Method of cultivation: 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 (approximately 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 10^4 to 10^5 cells/mL.

Test type:

static

Water media type:

other: The culture medium was prepared using reverse osmosis purified deionized water

Limit test:

no

Total exposure duration:

72 h

Hardness:

Not Specified

Test temperature:

24 ±1 ºC

pH:

7.5 ± 0.1

Nominal and measured concentrations:

The range-finding test
Nominal (mg/L): 0.10, 1.0, 10 and 100

The Definitive Test
Nominal (mg/L): 1.0, 3.2, 10, 32 and 100
Measured (geometric mean ) (mg/L): 0.35, 1.1, 4.6, 18 and 71

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type: closed with polyurethane foam stoppers
- Fill volume: 100 mL of test preparation
- Aeration: no
- Type: Static
- Initial cells nominal density: 5.00 x 10^3 cells per mL
- Control end cells density: 5.00 x 10^3 cells per mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
NaNO3: 25.5 mg/L
MgCl2.6H2O: 12.16 mg/L
CaCl2.2H2O: 4.41 mg/L
MgSO4.7H2O: 14.6 mg/L
K2HPO4: 1.044 mg/L
NaHCO3: 15.0 mg/L
H3BO3: 0.186 mg/L
MnCl2.4H2O: 0.415 mg/L
ZnCl2: 0.00327 mg/L
FeCl3.6H2O: 0.160 mg/L
CoCl2.6H2O: 0.00143 mg/L
Na2MoO4.2H2O: 0.00726 mg/L
CuCl2.2H2O: 0.000012 mg/L
Na2EDTA.2H2O: 0.30 mg/L
The culture medium was prepared using reverse osmosis purified deionized water and the pH adjusted to 7.5±0.1 with 0.1M NaOH or HCl.

TEST MEDIUM / WATER PARAMETERS
- 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 pH was measured using a Hach HQ30d Flexi handheld meter.
The temperature within the incubator was recorded daily.
The appearance of the test media was recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: yes
- Photoperiod: Continuous illumination
- Light intensity and quality: Approximately 7000 lux provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm.

TEST ORGANISM OBSERVATIONS
Samples were taken at 24, 51 and 72 hours and the cell densities determined using a haemocytometer and light microscope. Three determinations were made for each sample.
The nominal inoculated cell concentration (5.00 x 10^3 cells/mL) was taken as the starting cell density.
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
- Spacing factor for test concentrations: 3.2
- Range finding study: yes
- The range-finding test concentrations: Nominal (mg/L): 0.10, 1.0, 10 and 100. (Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.109 to 96.9 mg/L (95 to 109% of nominal concentrations). There was a significant decline in the measured test concentration at 72 hours in the range of 0.0114 to 51.5 mg/L (9 to 52% of nominal), indicating that the test item was unstable under test conditions.)
- The definitive test concentrations: (mg/L) Nominal : 1.0, 3.2, 10, 32 and 100; Measured: 0.35, 1.1, 4.6, 18 and 71. (Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.937 to 97.5 mg/L (93 to 98% of nominal concentrations). Analysis of the test preparations at 24, 48 and 72 hours showed a general decline in the measured test concentrations during the test to range from 0.771 to 99.6 mg/L at 24 hours and from 0.0481 to 56.1 mg/L at 48 hours and less than the limit of quantification (determined to be 0.0010 mg/L) to 26.4 mg/L (<1 to 26% nominal) at 72 hours, with the greatest losses occurring at the lower test concentrations. Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data. In cases where the measured concentration was less than the LOQ of the analytical method following current regulatory advice a value of half the LOQ (i.e. 0.00050 mg/L) was used to enable calculation of the geometric mean measured concentration)
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

30 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

It was not possible to calculate 95% confidence limits for the EC50 value as the data generated did not fit the models available for the calculation of confidence limits

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

4.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

18 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

12 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other:

Remarks:

95% Confidence Limits (mg/L): 9.6-16

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

4.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

18 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Details on results:

- Exponential growth in the control (for algal test): yes
- Times: Samples were taken at 24, 51 and 72 hours
- Observation of abnormalities: After 72 hours there were no abnormalities detected in the control or test cultures at 0.35, 1.1 and 4.6 mg/L; however some enlarged and misshapen cell were observed to be present in the test cultures at 18 and 71 mg/L.
- Any observations: At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72 Hour test period all control, 0.35, 1.1, 4.6 mg/L were observed to be bright green dispersions; the test cultures at 18 mg/L were observed to be green, slightly hazy dispersions and at 71 mg/L the cultures were observed to be hazy dispersions.

Results with reference substance (positive control):

- A positive control: Potassium dichromate (study number YJ31TQ), conducted between 12 November 2018 and 03 December 2018.
- Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 to 72 hour): 1.5 mg/L; 95% confidence limits 1.3 to 1.7 mg/L
EyC50 (0 to 72 hour): 0.76 mg/L; 95% confidence limits 0.69 to 0.85 mg/L

NOEC (growth rate): 0.50 mg/L
LOEC (growth rate): 1.0 mg/L
NOEC (yield): 0.50 mg/L
LOEC (yield): 1.0 mg/L
The results from the positive control with potassium dichromate were within the normal ranges for this reference item

Reported statistics and error estimates:

- One way analysis of variance incorporating Bartlett's test for homogeneity of variance;
- Dunnett's multiple comparison procedure;
- All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).
- For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerized interpolation using the Xlfit software package (IDBS). ECx values were then determined from the equation for the fitted line. Where appropriate 95% confidence limits for the EC50 values were calculated, using the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949).

Inhibition of Growth Rate

E_rC₁₀(0 to 72 hour): 15 mg/L

E_rC₂₀(0 to 72 hour): 20 mg/L

E_rC₅₀(0 to 72 hour): 30 mg/L;*

There were no statistically significant differences (P ≥0.05), between the control and 0.35, 1.1 and 4.6 mg/L test groups, however, all other test concentrations were significantly different (P<0.05) and therefore the NOEC based on growth rate was 4.6 mg/L. Correspondingly the LOEC based on growth rate was 18 mg/L.

*It was not possible to calculate 95% confidence limits for the EC₅₀value as the data generated did not fit the models available for the calculation of confidence limits

Inhibition of Yield

E_yC₁₀(0 to 72 hour): 2.4 mg/L

E_yC₂₀(0 to 72 hour): 4.3 mg/L

E_yC₅₀(0 to 72 hour): 12 mg/L; 95% confidence limits 9.6 to 16 mg/L

There were no statistically significant differences (P ≥0.05), between the control and 0.35, 1.1 and 4.6 mg/L test groups, however, all other test concentrations were significantly different (P<0.05) and therefore the NOEC based on yield was 4.6 mg/L. Correspondingly the LOEC based on yield was 18 mg/L.

Validation Criteria

·        The cell concentration of the control cultures must increase by a factor of at least 16 over the test period; actual achieved 356

·        The mean of the coefficients of variation of the section by section daily growth rates in the control cultures during the course of the test (Days 0 to 1, 1 to 2 and 2 to 3, for the 72‑Hour tests) must not exceed 35%; actual achieved 19%

·        The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7%; actual achieved 2%

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72 Hour period and based on the geometric mean measured test concentrations gave the following results:

Inhibition of Growth Rate
ErC10: 15 mg/L
ErC50: 30 mg/L;
NOEC: 4.6 mg/L
LOEC: 18 mg/L

Inhibition of Yield
EyC10: 2.4 mg/L
EyC50: 12 mg/L
NOEC: 4.6 mg/L
LOEC: 18 mg/L

Executive summary:

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The study was conducted according to OECD test guideline 201 and EC Method C.3, and in compliance with GLP. Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to aqueous solutions of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg/L for 72 hours. Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the geometric mean measured test concentrations:

Response Variable	EC50 (mg/L)	95% Confidence Limits (mg/L)			NOEC (mg/L)	LOEC (mg/L)
Growth Rate	30	Not determined*			4.6	18
Yield	12	9.6	-	16	4.6	18

*It was not possible to calculate 95% confidence limits for the EC₅₀value as the data generated did not fit the models available for the calculation of confidence limits

Description of key information

Inhibition of Growth Rate:

ErC10: 15 mg/L

ErC50: 30 mg/L; *

NOEC: 4.6 mg/L

LOEC: 18 mg/L

* It was not possible to calculate 95% confidence limits for the EC50 value as the data generated did not fit the models available for the calculation of confidence limits

Inhibition of Yield:

EyC10: 2.4 mg/L

EyC50: 12 mg/L; 95% confidence limits 9.6 to 16 mg/L

NOEC: 4.6 mg/L

LOEC: 18 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

30 mg/L

EC10 or NOEC for freshwater algae:

4.6 mg/L

Additional information

An algal growth inhibition study was conducted to determine the effect of Jasminlactone on the growth of Pseudokirchneriella subcapitata. The study was conducted according to OECD test guideline 201 and EC Method C.3, and in compliance with GLP.

 

Following a preliminary range-finding test, triplicate algal cultureswas exposed to aqueous solutions of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg/L 72 hours at a temperature of 24 ± 1 °C under static test conditions. Six replicate control cultures were also incubated concurrently with the test samples. Due to decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations, which were determined to be 0.35, 1.1, 4.6, 18 and 71 mg/L.

 

The growth rate and yield of Pseudokirchneriella subcapitatawere affected by the presence of Jasminlactone at measured test concentrations of 18 and 71 mg/L, the highest two concentrations tested. The NOEC and LOEC for both growth rate and yield were 4.6 and 18 mg/L, respectively.