Methyl 2-hexyl-3-oxocyclopentanecarboxylate

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

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)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

GLP compliance:

yes

Specific details on test material used for the study:

- Expiry date: 28 March 2009
- Description: Clear colourless liquid
- Test substance storage: At room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

During the final test singular samples for possible analysis were taken from all test concentrations and the control according to the following schedule: Frequency at t=0 h and t=72 h; Volume: 1.5 mL (22 mg/L) or 15 mL (others); Storage: Samples were stored in a freezer until analysis. At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling. Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at the highest substance concentration but without algae and samples for analysis were taken at the start of exposure and at the end of the test period. Additionally, singular reserve samples were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Vehicle:

no

Details on test solutions:

The standard test procedures required generation of test solutions, which contained completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that would disturb the test system was prevented as much as possible (e.g. film of the test substance on the water surface). The substance was completely soluble in test medium at the concentrations tested. Preparation of test solutions started with the highest test concentration being 100 mg/L (comb. limit/range-finding test) or 22 mg/L (final test). These concentrations were completely dissolved in test medium after overnight magnetic stirring. The lower test concentrations were consequently prepared by subsequent dilutions in test medium. The final test solutions were all clear and colourless. 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 104 cells/mL.

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata, strain: NIVA CHL 1
Source: In-house laboratory culture.
Reason for selection: This system is an unicellular algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol/L

Test temperature:

22.9 - 23.4°C

pH:

8.0 - 8.3

Nominal and measured concentrations:

Nominal: 1.0, 2.2, 4.6, 10, and 22 mg/L
Measured (t=0): 0.9, 2.1, 4.5, 8.9, 22.5 mg/L
Measured (t=72): 0.8, 1.7, 3.7, 8.4, 19.7 mg/L

Details on test conditions:

Fresh water algae culture:
- Stock culture: 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.
- Light intensity: 60 to 120 µE/m2/s when measured in the photosyntheticaily effective wavelength range of 400 to 700 nm.
- Stock culture medium: M1; according to the NPR 6505, formulated using Milli-Ro water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition: NaNO3: 500 mg/L; K2HPO4: 40 mg/L; MgSO4 7H2O: 76 mg/L; Na2CO3 10H2O: 54 mg/L; C6H807 H2O: 6 mg/L; NH4NO3: 330 mg/L; CaCl2 H2O: 36 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.
- Pre-culture: 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1E4 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: M2; according to the OECD 201 Guideline, formulated using Milli-Q water (tap water purified by reverse osmosis (milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges: Milli-Q water; Millipore Corp., Bedford, Mass., USA) preventing precipitation and with the following composition: NH4Cl: 15 mg/L; MgCl2 6H2O: 12 mg/L; CaCl2 2H2O: 18 mg/L; MgSO4 7H2O: 15mg/L; KH2PO4: 1.6 mg/L; FeCl3 6H2O 64 µg/L; Na2EDTA 2H2O: 100 µg/L, H3BO3: 185 µg/L; MnCl2 4H2O: 415 µg/L; ZnCl2: 3 µg/L; CoCl2 6H2O: 1.5 µg/L; CuCl2 2H2O: 0.01 µg/L; Na2MoO4 2H2O: 7 µg/L; NaHCO3: 50 mg/L.

Combined limit / range-finding test
- The project started with a combined limit/range-finding test. Six replicates of exponentially growing algae were exposed to a control and a concentration of 100 mg/L. Test procedure and conditions were similar to those applied in the final test with the following exceptions: Three replicates per concentration were exposed to 0.1, 1.0 and 10 mg/L; One extra test vessel per concentration without algae was used as background for the determination of the algal cell density at each time interval; pH was only measured in the control and the highest test concentration.

Final test:
- Controls: Test medium without test substance or other additives.
- Replicates: 3 replicates of each test concentration; 6 replicates of the control; 1 replicate of the highest concentration without algae.
- Test vessels: 100 mL, all-glass, containing 50 mL of test solution
- Medium: M2
- Cell density: An initial cell density of 1E4 cells/mL.
- Illumination: Continuously using TLD-lamps of the type 'Cool-white' of 30 Watt, with a light intensity within the range of 81 to 93 µE.m-2.s-1
- Incubation: Vessels were distributed at random in the incubator. During incubation the algal cells were kept in suspension by continuous shaking.
- Measurements: pH: At the beginning and at the end of the test. The pH of the solutions should preferably not deviate by more than 1.5 units during the test; Temperature of medium: Continuously in a temperature control vessel; Appearance of the cells: At the end of the final test microscopic observations were performed to verify a normal and healthy appearance of the inoculum culture and 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 cell densities were determined by spectrophotometric measurement of samples at 720 nm using a Varian Cary 50 single beam spectrophotometer with immersion probe (pathlength = 20 mm). Algal medium was used as blank.

Electronic data capture
- Observations/measurements in the study were recorded electronically using the following programme(s): Cary 50 single beam spectrophotometer including Cary UVVIS Pharma Upgrade Version 3.1 software (Varian, Mulgrave, Australia): Algal cell density. REES version 1.5 (comb. limit/range-finding) and REES Centron Environmental Monitoring system version SQL 2.0 (final test); (REES Scientific, Trenton, NJ, USA).

Data handling:
- Calibration curve: Quantification of cell densities was based on a calibration curve. Cell density was plotted versus extinction using spectrophotometric measurements of a minimum of six dilutions of an algal suspension with different cell densities. The calibration curve was composed using linear regression. The software automatically calculates the cell densities based on this curve for the spectrophotometric measurements at the various points in time during the test period.
- Determination of the NOEC and calculation of the EC50: For determination of the NOEC and the EC50 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 reduction of growth rate or inhibition of yield (ANOVA, Tukey test, Bonferroni t-test, TOXSTAT Release 3.5, 1996, D.D. Gulley, A.M. Boelter, H.L. Bergman). Additionally, the EC10 was determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993. Calculation of the EC50 and EC10 values was based on log-linear regression analysis of the percentages of growth rate reduction and the percentages of yield inhibition versus the logarithms of the corresponding nominal concentrations of the test substance.

Acceptability of the test:
1) In the controls, cell density increased by an average factor of > 16 within 2 days; 2) The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35%; 3) The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7%.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Analytical method:
- Calibration curves: Calibration curves were constructed using four concentrations. For each concentration, two responses were used. Linear regression analysis was performed using the least squares method with a 1/concentration^2 weighting factor. If necessary, a response was excluded from the curve since the back calculated concentration deviated > 10% from its nominal concentration. The coefficient of correlation (r) was > 0.99 for each curve.
- Procedural recovery samples: Mean recoveries of the procedural recovery samples were between 97% and 101%. Because the criterion that mean recoveries should be between 70% and 110% was met, the results for the test samples were accepted.
- Test samples: With the final test a small response at the retention time of the test substance was observed in the nominal 0 mg/L test samples. It was not considered to derive from the samples since a similar response was observed in the analytical blanks. The maximum contribution of the response to the test samples containing test substance was 0.3%.

Combined limit/range-finding test:
- Based on these results samples taken from nominal 1.0 and 10 mg/L were analysed. The initial measured concentrations were 0.8 and 9.3 mg/L, respectively. These concentrations remained stable during the test period (82-93% of initial). Therefore, the expected EC50 for growth rate reduction approximated 10 mg/L while the expected EC50 for yield inhibition was between 1.0 and 10 mg/L.

Final test
- Measured test substance concentrations: The actual test concentrations were in agreement with nominal at the start of the test with recoveries ranging between 86 and 102% relative to nominal. Measured concentrations at the end of the 72-hour test period showed that all concentrations had been maintained stable at more than 80% of the initial concentrations. Consequently, study results were based on nominal concentrations.
- Reduction of growth rate and inhibition of yield: Growth rates were in the range of the controls at 1.0 mg/L during the 72-hour test period, whereas the growth rate of algae exposed to 2.2 mg/L and higher were increasingly reduced. Statistically significant reduction of growth rate was found at test concentrations of 2.2 mg/L and higher (Bonferroni t and Tukey test, a = 0.05). Inhibition of yield increased with increasing concentration of test substance from 1.0 mg/L upwards resulting in more than 90% inhibition at and above nominally 10 mg/L. Statistically significant inhibition of yield was found at test concentrations of 2.2 mg/L and higher (Bonferroni t and Tukey test, a = 0.05). Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.

Validity criteria fulfilled:

yes

Description of key information

The measured 72h-ErC50 is 12 mg/L.

The measured NOErC is 1 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

12 mg/L

EC10 or NOEC for freshwater algae:

1 mg/L

Additional information

A Fresh Water Algal Growth Inhibition Test with the test substance is performed according to OECD guideline 201 and following GLP. A final test was performed based on the result of a preceding combined limit/range-finding test. Exponentially growing algal cultures (Pseudokirchneriella subcapitata) were exposed for 72 hours to a control and nominal test substance concentrations of 1.01, 2.21, 4.6, 10, and 22 mg/L. The initial algal cell density was 1E4 cells/mL and samples for analytical confirmation of actual exposure concentrations were taken at the start and the end of the test. Analyses showed that the actual test concentrations were in agreement with nominal at the start of the test with recoveries ranging between 86 and 102% relative to nominal. Measured concentrations at the end of the 72-hour test period showed that all concentrations had been maintained stable at more than 80% of the initial concentrations. Consequently, study results were based on nominal concentrations. The study met the acceptability criteria prescribed by the protocol and was considered valid. The test substance reduced growth rate of this fresh water algae species significantly at 2.2 mg/L and higher. The ECr50 for growth rate reduction was 12 mg/L with a 95% confidence interval ranging from 7.9 to 20 mg/L. The NOEC for growth rate reduction was 1.0 mg/L.

In addition, for the assessment of the aquatic toxicity of the test substance to algae, QSAR results were determined. ECOSAR v2.0 was used as the QSAR model and a water solubility of 186 mg/L and a partition coefficient of 3.2 was used for the prediction of the aquatic toxicity in algae. The results fell within the applicability domain of the QSAR model.