Cyclopentadecanone

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:

30 September 2016 to 9 October 2016

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)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000

Deviations:

not applicable

GLP compliance:

yes

Specific details on test material used for the study:

- Appearance: Colourless to very pale yellowish solid
- Test item storage: In refrigerator (2-8°C)
- Solubility in water: Insoluble
- Stability in water: Stable

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 800 µL samples for possible analysis were taken from all test concentrations and the control at t=0h, t=24h and t=72h.. In addition, the glasswool containing the undissolved residue was kept for possible analysis.
- Sampling method: At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
- Sample storage conditions before analysis: Not applicable, samples were transferred to the analytical laboratory at the Test Facility and analysed on the day of sampling.

Vehicle:

no

Details on test solutions:

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 means of filtration through a 0.45 μm membrane filter (RC55, Whatman) and used as the highest test concentration. Lower test concentrations 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. After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 1E+04 cells/mL. Any residual volumes were discarded.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light (60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm) in a climate room at a temperature of 21-24°C.

ACCLIMATION
- Culturing media and conditions: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tapwater purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition: NaNO3 500 mg/L; K2HPO4·3H2O: 52 mg/L; MgSO4·7H2O: 75 mg/L; Na2CO3·10H2O: 54 mg/L; C6H8O7·H2O: 6 mg/L; NH4NO3: 330 mg/L; CaCl2·2H2O: 35 mg/L; C6H5FeO7·xH2O: 6 mg/L;H3BO3: 2.9 mg/L; MnCl2·4H2O: 1.81 mg/L; ZnCl2: 0.11 mg/L; CuSO4·5H2O: 0.08 mg/L; (NH4)6Mo7O24·4H2O: 0.018 mg/L.
- Acclimation period: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1E+04 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg/L as CaCO3

Test temperature:

21 - 23 °C

pH:

8.1 - 8.2

Nominal and measured concentrations:

- Nominal concentrations: 0 (control), 1, 10 and 100% of a saturated solution prepared at a loading rate of 100 mg/L.
- Measured concentrations (TWA): n.d. 0.0028, 0.010, 0.17 mg/L.

See 'Any other information on materials and methods incl. tables' for more detail on measured concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass, containing 50 mL of test solution
- Type: closed
- Aeration: no
- Initial cells density: 1E+04 cells/mL
- Control end cells density: 162E+04 cells/mL
- No. of vessels per concentration: 3 for the 1.0 and 10% saturated solutions; 6 for the 100% saturated solution.
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: yes (M2, according to OECD 201 guideline).

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water (same as culture)
- Culture medium different from test medium: yes; M2 vs M1
- Intervals of water quality measurement: pH: at the beginning of the test; Temperature: continuously in a temperature control vessel

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Illumination: Continuously using TLD-lamps with a light intensity within the range of 85 to 93 μE·m-2·s-1

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (pathlength =20 mm). Algal medium was used as blank and the extra replicates, without algae, as background for the treated solutions. Cell densities were recorded at 24-hour intervals in the control and the highest test concentration. Lower concentrations were measured only at the end of the exposure period.
- Other: At the end of the test microscopic observations were performed on the highest test concentration to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Range finding study: yes (combined limit-range-finding test)
- Test concentrations: 0 (control), 1.0, 10 and 100% of a saturated solution prepared at a loading rate of 100 mg/L.
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate (September 2017)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.17 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.17 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: Statistically significant inhibition of growth rate was found to be <10% but considered biologically not relevant.

Details on results:

- Cell growth: Growth rates were in the range of the control at concentrations up to and including a TWA concentration of 0.010 mg/L during the 72-hour test period. Statistically significant inhibition of growth rate was found at the highest TWA concentration of 0.17 mg/L. However, inhibition was <10% and therefore considered biologically not relevant.
- Exponential growth in the control: yes
- Observation of abnormalities: No. Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 0.17 mg/L when compared to the control.
- Effect concentrations exceeding solubility of substance in test medium: No.

Results with reference substance (positive control):

- Results with reference substance valid: yes
- Dose-response test: yes (test concentrations: 0 (control), 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2mg/L)
- 72-h ErC50: 1.1 mg/L.
- Other: The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. Hence, the 72h-ErC50 for the algal culture tested corresponds with this range.

Reported statistics and error estimates:

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). The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany). No EC50-values could be calculated because the observed effects were below 50%.

Table: Growth Rate and Percentage Inhibition for the Total Test Period

TWA conc. (mg/L)	Growth rate
	Mean	Std. Dev.	n	% Inhibition
Control	1.696	0.0251	6	-
0.0028	1.700	0.0053	3	-0.3
0.010	1.684	0.0097	3	0.7
0.17	1.540	0.0652	6	9.2*

* Effect was statistically significant but biologically not relevant (<10%)

Validity criteria fulfilled:

yes

Remarks:

, see 'Overall remarks'.

Conclusions:

The 72h-ErC50 above, and the 72-h NOErC above or equal to the concentration obtained in a saturated solution prepared at 100 mg/L (0.17 mg/L based on TWA measured concentration).

Executive summary:

An algae toxicity test was performed according to OECD guideline No. 201 and in compliance with GLP criteria. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2000.

In a combined limit/range-finding test, replicates of exponentially growing algal cultures (initial cell density of 1E+04 cells/mL) were exposed to a control and to 1.0, 10 and 100% of the SS for 72 -hours. Samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure. At the start of the test, the actual measured test concentrations were 0.0097, 0.087 and 0.93 mg/L in 1.0, 10 and 100% of the SS, respectively. At the end of the 72-hour exposure period the initial concentrations of 0.0097 and 0.087 mg/L had decreased below the concentration of the lowest calibration solution (i.e. were below 0.004 mg/L). The initial concentration of 0.93 mg/L had decreased to 4.5% of initial at the end of the test. Based on these results, the Time Weighted Average (TWA) concentrations were calculated to be 0.0028, 0.010 and 0.17 mg/L and used to determine the NOEC and EC-values. Cell densities were recorded at 24-hour intervals in the control and the highest test concentration. Lower concentrations were measured only at the end of the exposure period.

Growth rates were in the range of the control at concentrations up to and including a TWA concentration of 0.010 mg/L during the 72-hour test period. Statistically significant inhibition of growth rate was found at the highest TWA concentration of 0.17 mg/L. However, inhibition was <10% and therefore considered biologically not relevant. In conclusion, due to the low solubility of test item in the test medium, concentration levels that might induce 50% inhibition of algal growth could not be reached. Hence, the 72h- EC50 for inhibition of growth rate (ErC50) was >0.17 mg/L. The 72h-NOEC for growth rate inhibition was <0.17 mg/L when based on statistical analysis and 0.17 mg/L when based on biological relevance.The study was valid.

Description of key information

An algae toxicity test was performed according to OECD guideline No. 201 and in compliance with GLP criteria. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2000.

In a combined limit/range-finding test, replicates of exponentially growing algal cultures (initial cell density of 1E+04 cells/mL) were exposed to a control and to 1.0, 10 and 100% of the SS for 72 -hours. Samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure. At the start of the test, the actual measured test concentrations were 0.0097, 0.087 and 0.93 mg/L in 1.0, 10 and 100% of the SS, respectively. At the end of the 72-hour exposure period the initial concentrations of 0.0097 and 0.087 mg/L had decreased below the concentration of the lowest calibration solution (i.e. were below 0.004 mg/L). The initial concentration of 0.93 mg/L had decreased to 4.5% of initial at the end of the test. Based on these results, the Time Weighted Average (TWA) concentrations were calculated to be 0.0028, 0.010 and 0.17 mg/L and used to determine the NOEC and EC-values. Cell densities were recorded at 24-hour intervals in the control and the highest test concentration. Lower concentrations were measured only at the end of the exposure period.

Growth rates were in the range of the control at concentrations up to and including a TWA concentration of 0.010 mg/L during the 72-hour test period. Statistically significant inhibition of growth rate was found at the highest TWA concentration of 0.17 mg/L. However, inhibition was <10% and therefore considered biologically not relevant. In conclusion, due to the low solubility of test item in the test medium, concentration levels that might induce 50% inhibition of algal growth could not be reached. Hence, the 72h- EC50 for inhibition of growth rate (ErC50) was >0.17 mg/L. The 72h-NOEC for growth rate inhibition was <0.17 mg/L when based on statistical analysis and 0.17 mg/L when based on biological relevance.The study was valid.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

0.17 mg/L