2-[10-(acetyloxy)-9a,11a-dimethyl-7-oxo-3H,3aH,3bH,4H,5H,7H,9aH,9bH,10H,11H,11aH-cyclopenta[a]phenanthren-1-yl]-2-oxoethyl acetate

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 January 2018 till 07 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23

Version / remarks:

December 14, 2000.

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-[10-(acetyloxy)-9a,11a-dimethyl-7-oxo-3H,3aH,3bH,4H,5H,7H,9aH,9bH,10H,11H,11aH-cyclopenta[a]phenanthren-1-yl]-2-oxoethyl acetate

EC Number:

607-277-2

Cas Number:

23825-05-0

Molecular formula:

C25H30O6

IUPAC Name:

2-[10-(acetyloxy)-9a,11a-dimethyl-7-oxo-3H,3aH,3bH,4H,5H,7H,9aH,9bH,10H,11H,11aH-cyclopenta[a]phenanthren-1-yl]-2-oxoethyl acetate

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: Off-white powder
- Storage condition of test material: At room temperature

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples for analysis were taken from all test concentrations with and without algae and the control at t=0 h, t=24 h and t=72 h.

Test solutions

Vehicle:

no

Details on test solutions:

The batch of PREDIAC-Z tested was not completely soluble in test medium at the loading rate initially prepared. Preparation of test solutions started with a loading rate of 100 mg/L applying a three-day period of magnetic stirring to ensure maximum dissolution of the test item in medium. Thereafter, the aqueous Saturated Solution (SS) was collected by filtration through a 0.45 μm membrane filter and used as the highest test concentration. Lower test concentrations (1% and 10% of the SS) were prepared by subsequent dilutions of the SS in test medium. All test solutions were clear and colorless at the end of the preparation procedure. Hence tested concentrations were PREDIAC-Z Solutions containing 1.0, 10 and 100% of a Saturated Solution prepared at a loading rate of 100 mg/L. As controls test medium without test item or other additives was used.

Test organisms

Test organisms (species):

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

Details on test organisms:

Species: Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), strain: NIVA CHL 1
Source: In-house laboratory culture.
Stock culture: Algae stock cultures were started by inoculating M1 growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C under light intensity 60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
Pre-culture: started 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium (M2 medium) at a cell density of 10000 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg CaCO3/L

Test temperature:

22-23°C

pH:

Range at start and end in control and 100 mg/L loading rate: 8.2-8.3

Nominal and measured concentrations:

Nominal concentrations were PREDIAC-Z Solutions containing 1.0, 10 and 100% of a Saturated Solution (SS) prepared at a loading rate of 100 mg/L. Samples taken from the control and the highest test concentration were analysed. At the start of the test, a concentration of 2.06 mg/L was measured in test samples (with algae) of the undiluted SS. This concentration declined to 1.34 and 1.35 mg/L after 24 and 72 hours, respectively in test samples (with algae). Based on these results, the effect parameters were based on the Time Weighted Average (TWA) exposure concentration, calculated to be 1.4 mg/L in the undiluted SS.
The concentrations measured in the samples without algae were comparable to the corresponding results of samples with algae (2.11, 1.48 and 1.15 mg/L at t=0, t=24 and t=72 hours, respectively). It should be noted that small responses were detected in the control throughout the test. These responses were probably caused by carry over at the start of the test and after 24 hours of exposure, since similar responses were found in the analytical blanks. At the end of the test, the measured response was unlikely to be caused by carry over alone. The source of the increased response is unknown but considering that the concentration could be estimated only by extrapolation of the calibration curve and was 0.033% of the concentration measured at the highest test concentration, this was considered negligible.

Details on test conditions:

Replicates: 6 replicates each of the control and the highest test concentration, 3 replicates of each intermediate test concentration, 1 extra replicate of each test group for sampling purposes after 24 hours of exposure, 1 or 2 replicates of each test concentration without algae.
Test vessels: 100 mL, all-glass, containing 50 mL of test solution.
Medium: M2.
Cell density: An initial cell density of 10000 cells/mL.
Illumination: Continuously using TLD-lamps with a light intensity within the range of 87 to 89 μE.m-2.s-1.
Incubation: Capped vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.
pH: measured at the beginning and at the end of the test for the control and the highest test concentration.
Temperature of medium: measured continuously in a temperature control vessel.
Appearance of the cells: at the end of the final test microscopic observations were performed on the highest test concentration to observe for any abnormal appearance of the algae.
Recording of Cell Densities: at the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter (24, 48 and 72 hours), cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 10 mm). Algal medium was used as blank and the extra replicates, without algae, as background for the treated solutions.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 1.4 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: See remark.

Remarks:

The effect parameters were based on the measured TWA concentration in the filtrate of a Saturated Solution being considered the maximum soluble concentration of the test item in test medium.

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 1.4 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: See remark

Remarks:

The effect parameters were based on the measured TWA concentration in the filtrate of a Saturated Solution being considered the maximum soluble concentration of the test item in test medium.

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1.4 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: See remark

Remarks:

The effect parameters were based on the measured TWA concentration in the filtrate of a Saturated Solution being considered the maximum soluble concentration of the test item in test medium.

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 1.4 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: See remark.

Remarks:

The effect parameters were based on the measured TWA concentration in the filtrate of a Saturated Solution being considered the maximum soluble concentration of the test item in test medium. A statistically significant reduction of growth rate of 0.75% was not considered biologically relevant.

Details on results:

In the PREDIAC-Z Solutions containing 1.0, 10 and 100% of a Saturated Solution prepared at a loading rate of 100 mg/L, the % inhibition of growth rate compared to the control after 72 hours was -1.1%, -0.48% and -0.75%, respectively. Only the inhibition of 0.75% observed in the highest test concentration was statistically significant. Since the effect (0.75%) was well below 10% it was considered to be biologically not relevant and the NOEC for growth rate inhibition was set at 1.4 mg/L. Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the limit concentration when compared to the control.

Results with reference substance (positive control):

Potassium dichromate (K2Cr2O7) was tested on Pseudokirchneriella subcapitata (strain: NIVA CHL-1) in a separate test on 02-05 January 2018. The EC50 for growth rate inhibition (72h-ERC50) was 0.86 mg/L with a 95% confidence interval ranging from 0.84 to 0.88 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. The observed 72h-ERC50 for the algal culture tested corresponds with this range.

Reported statistics and error estimates:

For determination of the NOEC the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Two-sample t-test Procedure, α=0.05, one-sided, smaller). No EC10 or EC50-values could be calculated because the test item proved to be non-toxic (EC50 > maximum soluble concentration tested). The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Any other information on results incl. tables

The validity criteria of OECD 201 (2011) were satisfied:

1.      In the control, cell density increased by an average factor of at least 16 within the exposure period (i.e. 225).

2.      The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35% (i.e. 10%).

3.      The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7% (i.e. 0.89%).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See above under "Any information on results inc. tables".

Conclusions:

Under the conditions of the present study with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), no biologically relevant inhibition of growth rate was recorded at any of the concentrations of PREDIAC-Z tested. The EC50 for growth rate inhibition (72h-ERC50) was beyond the range tested, i.e. exceeded a measured concentration of 1.4 mg/L being considered the maximum soluble concentration of the test item in test medium. The 72h-NOEC for growth rate inhibition was 1.4 mg/L.

Executive summary:

The objective of the study was to evaluate PREDIAC-Z for its ability to generate toxic effects in Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) during an exposure period of 72 hours and, if possible, to determine the NOEC, EC₁₀and EC₅₀ for inhibition of growth rate (and inhibition of yield, not further considered however).

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2000.

A Saturated Solution (SS) was prepared at a loading rate of 100 mg/L and used as the highest concentration. Lower concentrations (1% and 10% of SS) were prepared by diluting the highest concentration in test medium.

A combined limit/range-finding test was performed. Six exponentially growing algal cultures per group were exposed to an untreated control and to the undiluted SS in the limit test. In addition, three replicates per group were exposed to 1.0 and 10% of the SS in a combined range-finding test. The initial algal cell density was 10⁴cells/mL.The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start and after 24 and 72 hours of exposure.

Samples taken from the undiluted SS were analysed. At the start of the test, the actual test concentration in the undiluted SS was 2.1 mg/L. This concentration declined to 1.34 and 1.35 mg/L after 24 and 72 hours, respectively. Based on these results, the Time Weighted Average (TWA) exposure concentration was calculated to be 1.4 mg/L in the undiluted SS and used to determine the effect parameters.

The study met the validity criteria of OECD 201 and was considered valid.

Under the conditions of the present study, no biologically relevant inhibition of growth rate was recorded at any of the concentrations of PREDIAC-Z tested. The EC10, EC20 and EC50 for growth rate inhibition was beyond the range tested, i.e. exceeded a measured concentration of 1.4 mg/L being considered the maximum soluble concentration of the test item in test medium. The 72h-NOEC for growth rate inhibition was 1.4 mg/L.