Reaction mass of (E)-1-(3,6-dimethylcyclohex-3-en-1-yl)-2-methylpent-1-en-3-one and (E)-1-(4,6-dimethylcyclohex-3-en-1-yl)-2-methylpent-1-en-3-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:

11 February 2019 - 01 June 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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Identification: PG-RAW-90-032
- Physical State/Appearance: Clear colourless liquid
- Storage Conditions: Approximately 4 °C in the dark

Analytical monitoring:

yes

Details on sampling:

- Sample storage conditions before analysis: frozen before analysis
- Sampling times: 24, 46 and 72 hours (Three determinations were made for each sample)

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- The results obtained from the preliminary media preparation trial indicated that a dissolved test item concentration of approximately 10.8 mg/L could be obtained in ASTM algal culture medium using a saturated solution method of preparation.
- An initial range-finding test was conducted taking no precautionary measures with regards to the volatile nature of the test item. After performing a volatility trial, a second range-finding test was conducted in which the test design was modified for the testing of a volatile substance.
- Following preliminary range finding tests, an initial experiment was conducted at nominal test concentrations of 0.10, 0.32, 1.0, 3.2 and 10% v/v saturated solution. Due to insufficient inhibition of growth, the was considered to be inadequate.
- The following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100% v/v saturated solution.

- Experimental Preparation: A nominal amount of test item (1100 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 liters used to pre condition the filter was discarded) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give stock solutions of 1.0, 3.2, 10 and 32% v/v saturated solution. An aliquot (1800 mL) of each of the stock solutions was separately inoculated with 12.5 mL of algal suspension to give the required test concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution. The stock solutions and each prepared concentration 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
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland
- Initial cell density of approximately 10^3 cells/mL.
- Age of inoculum (at test initiation): Not specified
- Master cultures: were maintained in the laboratory by the periodic replenishment of culture medium (Section 3.3). The master cultures were kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C.

ACCLIMATION
- Acclimation period: Not specified

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

Remarks on exposure duration:

N/A

Post exposure observation period:

N/A

Hardness:

Not specified

Test temperature:

24 ±1 °C

pH:

7.9 - 9.1

The pH value of the control cultures was observed to increase from pH 8.0 at 0 hours to pH 9.2 at 72 hours.
The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guideline.

Dissolved oxygen:

Not specified

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

2nd range finding test:
Nominal test concentrations of 0.10, 1.0, 10 and 100% v/v

Definitive test:
Initial Test: Nominal concentrations: 0.10, 0.32, 1.0, 3.2 and 10% v/v saturated solution (results not reported as insufficient inhibition of growth was observed)
Second Test: Nominal concentrations: 1.0, 3.2, 10, 32 and 100% v/v saturated solution
Second Test: Measured concentrations (mg/L): 0.09, 0.28, 0.83, 2.7 and 6.7.
At 24 hours measured concentrations were in the range of 0.010 to 7.0 mg/L (91% to 85% of the 0 Hour measured test concentrations),
At 48 hours measured concentrations were in the range of0.08 to 6.6 mg/L (73% to 81% of the 0 Hour measured test concentrations) and
At 72 hours measured concentrations were in the range of0.081 and 5.0 mg/L (61% to 71% of the 0 Hour measured test concentrations.
The average geometric mean measured test concentrations for the 0, 24, 48 and 72 hour values were determined to be 0.09, 0.28, 0.83, 2.7 and 6.7 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks completely filled with test preparation
- Type (delete if not applicable): Sealed with ground glass stoppers to minimize the potential for losses of the test item due to volatilization
- Aeration: no
- Type of flow-through: static
- Renewal rate of test solution: static
- Initial nominal cells density: 5.00 x 10^3 cells/ mL
- Number of replicates per test culture: 3
- Number of replicates in control: 6

TEST MEDIUM / WATER PARAMETERS
- NaNO3L: 25.5
- MgCl2.6H2O: 12.16
- CaCl2.2H2O: 4.41
- MgSO4.7H2O: 14.6
- K2HPO4: 1.044
- NaHCO3: 15.0
- H3BO3: 0.186
- MnCl2.4H2O: 0.415
- ZnCl2: 0.00327
- FeCl3.6H2O: 0.160
- CoCl2.6H2O: 0.00143
- Na2MoO4.2H2O: 0.00726
- CuCl2.2H2O: 0.000012
- Na2EDTA.2H2O: 0.30
- The culture medium was prepared using reverse osmosis purified deionized water
- The culture medium used for the tests was prepared with the addition of 250 mg/L of sodium bicarbonate to prevent inhibition of growth due to the restriction in gaseous exchange associated with conducting the test in an enclosed system.

OTHER TEST CONDITIONS
- Sterile test conditions: not specified
- Adjustment of pH: yes to 7.5 ± 0.1 with 0.1N NaOH or HCl.
- Photoperiod: Constant illumination
- Light intensity and quality: approximately 7000 lux, provided by warm white lighting (380 to 730 nm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Sampling times: 24, 46 and 72 hours (Three determinations were made for each sample)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: yes,
- Test concentrations: 0.10, 0.32, 1.0, 3.2 and 10% v/v saturated solution
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 2.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.83 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 2.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 1.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.83 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 2.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Observations on Cultures
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 0.09, 0.28, 0.83 and 2.7 mg/L, however some misshapen cells were observed in the test cultures at 6.7 mg/L.

Observations on Test Item Solubility
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, 0.09, 0.28 and 0.83 mg/L test cultures were green dispersions and the 2.7 and 6.7 mg/L test cultures were clear colorless solutions.

Results with reference substance (positive control):

A positive control (Envigo study number YJ31TQ) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.
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 based on growth rate: 0.50 mg/L
NOEC based on yield: 0.50 mg/L
LOEC based on growth rate: 1.0 mg/L
LOEC based on yield: 1.0 mg/L

Reported statistics and error estimates:

Xlfit software package (IDBS).
ECx values were then determined from the equation for the fitted line.
One way analysis of variance incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparison procedure for comparing several treatments with a control was carried out on the growth rate and yield data after 72 hours to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

 Inhibition of Growth Rate

E_rC₁₀(0 to 72 hour): 1.4 mg/L

E_rC₂₀(0 to 72 hour): 1.8 mg/L

E_rC₅₀(0 to 72 hour): 2.9 mg/L; 95% confidence limits 0.66 to 12 mg/L

There were no statistically significant differences (P≥0.05), between the control and 0.09, 0.28 and 0.83 mg/L test groups; however, all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on growth rate was 0.83 mg/L. Correspondingly the LOEC based on growth rate was 2.7 mg/L.

 Inhibition of Yield

E_yC₁₀(0 to 72 hour): 0.99 mg/L

E_yC₂₀(0 to 72 hour): 1.2 mg/L

E_yC₅₀(0 to 72 hour): 1.6 mg/L

It was not possible to calculate the 95% confidence limits as the data generated did not fit the model available.

There were no statistically significant differences (P ≥ 0.05), between the control and 0.09, 0.28 and 0.83 mg/L test groups; however, all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on growth rate was 0.83 mg/L. Correspondingly the LOEC based on growth rate was 2.7 mg/L.

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:
Growth Rate:
EC50 (mg/L): 2.9
95% Confidence Limits (mg/L): 0.66 - 12
NOEC (mg/L): 0.83
LOEC (mg/L): 2.7

Yield:
EC50 (mg/L): 1.6
It was not possible to calculate the 95% confidence limits as the data generated did not fit the model available.
NOEC (mg/L): 0.83
LOEC (mg/L)L 2.7

Executive summary:

The effect of PG-RAW-90-032 on the growth of Pseudokirchneriella subcapitata has been investigated (Covance CRS, 2019, JX07FY). The method followed the OECD test Guideline 201 and was conducted in compliance with GLP.

Following preliminary range finding tests, and an initial experiment, Pseudokirchneriella subcapitata was exposed to solutions of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution in triplicates for 72 hours. After 72 hours the measured concentrations were in range of 0.081 and 5.0 mg/L (61% to 71% of the 0 Hour measured test concentrations). The test was performed under constant illumination and shaking at a temperature of 24 ±1 °C under closed and static conditions. 

After 72 hours of exposure there were no abnormalities observed in the control or test cultures at 0.09, 0.28, 0.83 and 2.7 mg/L, however some misshapen cells were observed in the test cultures at 6.7 mg/L. The EC50 were 2.9 and 1.6 mg/L for growth rate and yield, respectively. No observed effect concentration was 0.83 mg/L and lowest observed effect concentration was 2.7 mg/L for both growth rate and yield.

Description of key information

Growth Rate:

EC50 (mg/L):  2.9

95% Confidence Limits (mg/L): 0.66 - 12

NOEC (mg/L): 0.83

LOEC (mg/L): 2.7

Yield:

EC50 (mg/L): 1.6

It was not possible to calculate the 95% confidence limits as the data generated did not fit the model available.

NOEC (mg/L): 0.83

LOEC (mg/L)L 2.7

Key value for chemical safety assessment

EC50 for freshwater algae:

2.9 mg/L

EC10 or NOEC for freshwater algae:

0.83 mg/L

Additional information

The effect of PG-RAW-90-032 on the growth of Pseudokirchneriella subcapitatahas been investigated (Covance CRS, 2019, JX07FY). The method followed the OECD test Guideline 201 and was conducted in compliance with GLP.

Following preliminary range finding tests, and an initial experiment, Pseudokirchneriella subcapitata was exposed to solutions of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution in triplicates for 72 hours. After 72 hours the measured concentrations were in range of 0.081 and 5.0 mg/L (61% to 71% of the 0 Hour measured test concentrations). The test was performed under constant illumination and shaking at a temperature of 24 ±1 °C under closed and static conditions. 

After 72 hours of exposure there were no abnormalities observed in the control or test cultures at 0.09, 0.28, 0.83 and 2.7 mg/L, however some misshapen cells were observed in the test cultures at 6.7 mg/L. The EC50 were 2.9 and 1.6 mg/L for growth rate and yield, respectively. No observed effect concentration was 0.83 mg/L and lowest observed effect concentration was 2.7 mg/L for both growth rate and yield.