Reaction mass of 4-methyl-1-oxaspiro[5.5]undec-3-ene and 4-methyl-1-oxaspiro[5.5]undec-4-ene and 4-methylidene-1-oxaspiro[5.5]undecane

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 Oct 2017 - 08 Dec 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, 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:

no

GLP compliance:

yes

Specific details on test material used for the study:

- Solubility in water: 6300.4 mg/L at 24°C
- Stability in water: Not indicated

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all test concentrations and control. In addition, compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate item concentration but without algae.
- Frequency: at the start of the test, and after 24 hours, 48 hours, and 72 hours.
- Sampling method: 15 mL samples were taken. At the end of the exposure period, the replicates with algae were not pooled at each concentration before sampling. Instead, samples were taken from replicate 1 of each test concentration.
- Sample storage conditions before analysis: samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Preparation of test solutions started with loading rates individually prepared at 1.0 to 100 mg/L. A 60- to 71-minutes period of magnetic stirring was applied to ensure maximum dissolution of the test item in test medium. The obtained mixtures were allowed to settle for a period of 52-56 minutes. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning and used as test concentrations. All test solutions were clear and colorless at the end of the preparation procedure. After preparation, volumes of 40 mL were added to each replicate vessel of the respective test concentration containing a 0.8 mL of an algal suspension 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.
- Age of inoculum (at test initiation): 3 days before start of the test (pre-culture)

CULTIVATION
- Method: 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 in a climate room at a temperature of 21 - 24°C.
- Stock culture medium: M1 (according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”))
- Pre-culture: 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.
- Pre-culture medium: Adjusted M2 (according to the OECD 201 Guideline)
- Light intensity: 60 to 120 μE/m^2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.

ACCLIMATION
- Acclimation period: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg/L expressed as CaCO3

Test temperature:

22 - 23 °C

pH:

7.4 - 8.1

Nominal and measured concentrations:

- Nominal concentrations: WAFs individually prepared at loading rates of 1.0, 3.2, 10, 32 and 100 mg/L and a blank control.
- Measured concentrations (average*): 0.25, 2.0, 7.8, 26 and 37 mg/L.

* For more detail on nominal versus measured concentrations, see 'Any other information on materials and methods'.

Details on test conditions:

TEST SYSTEM
- Test vessel: 40 mL, glass vials, airtight closed, with no headspace to prevent any loss of test item due to volatilization, containing 40 mL of test solution
- Initial cells density: 1E+04 cells/mL
- Control end cells density: 2.73E+06 cells/mL
- No. of vessels per concentration: 3
- No. of vessels per control: 6
- 2 extra replicates of each test group for sampling purposes after 24 and 48 hours of exposure
- 1 or 3 replicates of each test concentration without algae.

GROWTH MEDIUM
- Standard medium used: yes, adjusted M2

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: yes, M1 (according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) stock culture medium versus Adjusted M2 (according to the OECD 201 Guideline) test medium.
- Intervals of water quality measurement: pH at the beginning and at the end of the test, temperature of medium continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no
- Adjustment of pH: no
- Illumination: Continuously using TLD-lamps with a light intensity within the range of 72 to 79 µE·m-2·s-1.

EFFECT PARAMETERS MEASURED:
- Effect parameters: cell densities at t=0h and at 24, 48 and 72 hours after start of the test.
- 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 cuvettes (path length = 10 mm). Algal medium was used as blank and the extra replicates, without algae, as background for the treated solutions.
- Other: Appearance of the cells at the end of the final test, microscopic observations were performed on the control and the WAF prepared at 10 mg/L to observe for any abnormal appearance of the algae.

RANGE-FINDING STUDY
- Test concentrations: WAFs prepared at loading rates of 1, 10 and 100 mg/L and a blank control.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (performed Sep 2017)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: 95% C.I.: 10 - 12 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: 95% C.I.: 2.8 - 3.6 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Details on results:

- Observations: Growth rates were in the range of the controls at the two lowest test concentrations during the 72-hour test period, whereas the growth rate of algae exposed to 7.8 mg/L and higher were increasingly reduced. Statistically significant inhibition of growth rate was found at the three highest test concentrations. Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 7.8 mg/L when compared to the control.
- Exponential growth in the control: yes (factor 273 increase in controls)
- Observation of abnormalities: No
- Aggregation of algal cells: No
- Other: The inhibition of algal growth observed at 0.25 mg/L fell outside the dose-related effect gradient. The reason for this is not clear. The concentrations measured throughout the test, and the cell densities measured after 24 hours of exposure fell within of what could be expected in relation to the other test groups. It was consequently considered that the effects observed later during the test were not dose-related and the lowest test concentration was thus removed from the calculation of the effect concentrations to improve model performance and obtain more realistic and representative results.
- Any indication that effect concentrations exceeded the solubility of substance in test medium: No.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: 72-h ErC50 = 1.1 mg/L (95% C.I.: 1.1 - 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:

- Statistical significance: 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 (Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm, α=0.05, one-sided, smaller).
- ECx values: calculations based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding average exposure concentrations of the test item. The effects observed at the lowest test concentration were considered to not be dose-related and removed for the calculation of the ECx values to improve the model performance and obtain more realistic results. The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

DETAIL ON RESULTS

Table: Growth rate and percentage inhibition for the total test period

Average conc. (mg/L)	Mean	Std Dev.	n	% inhibition
0 (control)	1.870	0.0163	6	--
0.25	1.805	0.0257	3	3.4*
2.0	1.852	0.0126	3	0.93
7.8	1.591	0.0136	3	15 **
26	0.130	0.2249	3	93 **
37	0.062	0.1080	3	97 **

* Effect considered as not dose-related

** Effect was statistically significant

Validity criteria fulfilled:

yes

Conclusions:

The 72-h ErC50 and ErC10 values are 11 mg/L and 3.2 mg/L, respectively in the freshwater green algae Pseudokirchneriella subcapitata. The NOErC is 2.0 mg/L.

Executive summary:

The algae toxicity was examined in a study according to OECD TG 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.

Exponentially growing algal cultures at an initial algal cell density of 1E+04 cells/mL, were exposed to an untreated control, and to WAFs prepared at 1.0, 3.2, 10, 32 and 100 mg/L test item for 72 hours under static conditions. To prevent any loss of test item due to volatilization, the headspace in the test vessels was reduced to a minimum and the test vessels closed airtightly. Samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24, 48 and 72 hours of exposure. The concentrations measured at the start of the test were 0.42, 2.6, 12, 32 and 48 mg/L, respectively. These concentrations decreased to 27-59% of the initial concentrations during the exposure period. Based on these results, the geometric mean average exposure concentrations were calculated to be 0.25, 2.0, 7.8, 26 and 37 mg/L. The study met the acceptability criteria prescribed by the study plan and was considered valid.

Growth rates were in the range of the controls at the two lowest test concentrations during the 72-hour test period, whereas the growth rate of algae exposed to 7.8 mg/L and higher were increasingly reduced. Statistically significant inhibition of growth rate was found at the three highest test concentrations: 7.8, 26 and 37 mg/l. A dose-related increase of inhibition of growth rate was observed at the end of the test, resulting in 15, 97 and 100% inhibition, respectively. The inhibition of algal growth observed at 0.25 mg/L was considered to not be dose-related and was removed from the calculation of the effect concentrations. Based on these findings, the 72 -h ErC50 and ErC10 values were determined at 11 and 3.2 mg/L, respectively. The NOErC was determined at 2.0 mg/L.

Description of key information

The algae toxicity was examined in a study according to OECD TG 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.

Exponentially growing algal cultures at an initial algal cell density of 1E+04 cells/mL, were exposed to an untreated control, and toWAFs prepared at 1.0, 3.2, 10, 32 and 100 mg/L test item for 72 hours under static conditions. To prevent any loss of test item due to volatilization, the headspace in the test vessels was reduced to a minimum and the test vessels closed airtightly. Samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24, 48 and 72 hours of exposure.The concentrations measured at the start of the test were 0.42, 2.6, 12, 32 and 48 mg/L, respectively. These concentrations decreased to 27-59% of the initial concentrations during the exposure period. Based on these results, the geometric mean average exposure concentrations were calculated to be 0.25, 2.0, 7.8, 26 and 37 mg/L.The study met the acceptability criteria prescribed by the study plan and was considered valid.

Growth rates were in the range of the controls at the two lowest test concentrations during the 72-hour test period, whereas the growth rate of algae exposed to 7.8 mg/L and higher were increasingly reduced. Statistically significant inhibition of growth rate was found at the three highest test concentrations: 7.8, 26 and 37 mg/l. A dose-related increase of inhibition of growth rate was observed at the end of the test, resulting in 15, 97 and 100% inhibition, respectively. The inhibition of algal growth observed at 0.25 mg/L was considered to not be dose-related and was removed from the calculation of the effect concentrations. Based on these findings, the 72 -h ErC50 and ErC10 values were determined at 11 and 3.2 mg/L, respectively. The NOErC was determined at 2.0 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

11 mg/L

EC10 or NOEC for freshwater algae:

3.2 mg/L