Oxacycloheptadecan-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:

7 October 2016 to 15 December 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:

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.

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. The obtained aqueous mixture was allowed to settle for a period of 20-35 minutes. Thereafter, the Saturated Solution (SS) was collected by means of siphoning through glass wool and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. Test solutions up to and including 18% of the SS were clear and colorless at the end of the preparation procedure. Test solutions ranging from 32 to 100% of the SS were increasingly hazy. The SS prepared for the combined limit/rangefinding test was observed to be very slightly yellow. 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.

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:

Start: 7.8 - 8.2
End: 8.4 - 8.5

Nominal and measured concentrations:

- Nominal concentrations: 0, 10, 18, 32, 56 and 100% of a saturated solution prepared at a loading rate of 100 mg/L.
- Measured concentrations: all analyzed concentrations were <0.004 mg/L (below the concentration of the lowest calibration solution)

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: 194E+04 cell/mL
- No. of vessels per concentration: 3
- 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 88 to 91 μE·m-2·s-1

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Cells were counted using a microscope and a counting chamber.
- Other: At the end of the test microscopic observations were performed on all test groups to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Range finding study: yes
- 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.:

> 0.004 mg/L

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

No effects on growth rate observed up to the concentration obtained in a saturated solution prepared at a loading rate of 100 mg/L. This concentration was above the solubility limit in test medium but could not be measured because it was below the concentration of the lowest calibration solution (i.e. below 0.004 mg/L).

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.004 mg/L

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: NOErC = max. solubility of test substance in test medium.

Details on results:

- Growth rate: No significant differences were recorded between the values for growth rate or yield at any of the test concentrations when compared to the control group.
- 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 all test concentrations when compared to the control.
- Effect concentrations exceeding solubility of substance in test medium: From 24 hours onwards the replicates containing 56 and 100% of the saturated solution were observed to be slightly hazy and hazy, respectively, indicating that the actual test concentrations were above the solubility limit in test medium.

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 3.2 mg/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 dilutions of the SS compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Dunnett`s Multiple t-test Procedure, α=0.05, one-sided, smaller). The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

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

Test conc. as %SS at 100 mg/L	Growth rate
	Mean	Std. Dev.	n	% inhibition
Control	1.754	0.0397	6	--
10	1.756	0.0155	3	-0.1
18	1.750	0.0587	3	0.2
32	1.772	0.0398	3	-1.1
56	1.775	0.0231	3	-1.0
100	1.731	0.0149	3	1.3

 

Validity criteria fulfilled:

yes

Remarks:

see 'Overall remarks'

Conclusions:

The 72-h ErC50 and the 72-h NOErC were above the concentration obtained in a saturated solution prepared at 100 mg/L. This concentration was above the solubility limit of the substance in test medium (as indicated by the haziness) but could not be measured because it was below the concentration of the lowest calibration solution (i.e. below 0.004 mg/L).

Executive summary:

An algae toxicity 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.

The test item was not completely soluble in test medium at the loading rate initially prepared. A final test was performed based on the results of a preceding combined limit/range-finding test. A Saturated Solution (SS) was prepared at a loading rate of 100 mg/L and used as the highest concentration. Lower concentrations were prepared by diluting the SS in test medium. Six replicates of exponentially growing algal cultures were exposed to a control, whereas three replicates per group were exposed to 10, 18, 32, 56 and 100% of the SS. The initial algal cell density was 1.0E+04 cells/mL and the total exposure period was 72 hours. Samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure. From 24 hours onwards the replicates containing 56 and 100% of the SS were observed to be slightly hazy and hazy, respectively, indicating that the actual test concentrations were above the solubility limit in test medium. Samples taken from 1.0, 10 and 100% of the SS, with and without algae, were analysed and the actual measured concentrations were all below the concentration of the lowest calibration solution (i.e. were below 0.004 mg/L) from the start. Algae cell densities were counted at 24, 48 and 72 hours after start of exposure and growth rates and percentage inhibition of growth rates calculated. The study met the acceptability criteria and was considered valid.

No significant differences were recorded between the values for growth rate of any of the test concentrations when compared to the control group. Due to the low solubility of the test item in test medium, concentration levels that might induce statistically significant inhibition of algal growth could not be reached. The 72h-NOEC and the 72h-EC50 for inhibition of both growth rate and yield were above the concentration obtained in a SS prepared at 100 mg/L. This concentration was above the solubility limit of the substance in test medium but could not be measured because it was below the concentration of the lowest calibration solution (i.e. below 0.004 mg/L).

Description of key information

An algae toxicity 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.

The test item was not completely soluble in test medium at the loading rate initially prepared. A final test was performed based on the results of a preceding combined limit/range-finding test. A Saturated Solution (SS) was prepared at a loading rate of 100 mg/L and used as the highest concentration. Lower concentrations were prepared by diluting the SS in test medium. Six replicates of exponentially growing algal cultures were exposed to a control, whereas three replicates per group were exposed to 10, 18, 32, 56 and 100% of the SS. The initial algal cell density was 1.0E+04 cells/mL and the total exposure period was 72 hours. Samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure. From 24 hours onwards the replicates containing 56 and 100% of the SS were observed to be slightly hazy and hazy, respectively, indicating that the actual test concentrations were above the solubility limit in test medium. Samples taken from 1.0, 10 and 100% of the SS, with and without algae, were analysed and the actual measured concentrations were all below the concentration of the lowest calibration solution (i.e. were below 0.004 mg/L) from the start. Algae cell densities were counted at 24, 48 and 72 hours after start of exposure and growth rates and percentage inhibition of growth rates calculated. The study met the acceptability criteria and was considered valid.

No significant differences were recorded between the values for growth rate of any of the test concentrations when compared to the control group. Due to the low solubility of the test item in test medium, concentration levels that might induce statistically significant inhibition of algal growth could not be reached. The 72h-NOEC and the 72h-EC50 for inhibition of both growth rate and yield were above the concentration obtained in a SS prepared at 100 mg/L. This concentration was above the solubility limit of the substance in test medium but could not be measured because it was below the concentration of the lowest calibration solution (i.e. below 0.004 mg/L).