1,1,2,3,3,6-hexamethylindan-5-yl methyl ketone

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 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes

Specific details on test material used for the study:

- Appearance: Off-white powder
- Storage conditions: At room temperature in the dark
- Stability: Stable for at least 12 months under storage conditions

Analytical monitoring:

yes

Details on sampling:

The concentration of the test substance in the samples was determined by means of gas chromatography with flame ionization detection. Samples (ca. 20 mL) were withdrawn from the control, lowest , middle and highest concentration tested. Samples from the other concentrations were also withdrawn and stored for additional analyses, when needed. The concentration of the test substance in the solution withdrawn directly from the stock solution was also determined. The samples were stored in the refrigerator in the dark until analysis. All samples will be taken in single-fold and analyzed in duplicate. Samples were taken at 0, 24, 48 and 72 hours of testing. The initial samples for analysis (0 hours) were withdrawn from the vessels after addition of the test organisms and stabilization of the test system. The samples were stored for a maximum period of 4 weeks.

Vehicle:

no

Details on test solutions:

A test substance stock solution was prepared by weighing out the test substance (0.05000 gram) using a analytical balans (Sartorius, 160P, Breukelen, The Netherlands). This was transferred to a separatory funnel filled with 500 mL test medium. This mixture was ultrasonically treated for approximately 20 minutes and subsequently mixed for approximately 20 hours at room temperature using a magnetic stirrer. Thereafter, the liquid was filtered using a glass fibre filter (Schleicher& Schull, GF 92 Glasfaser filter (ref.nr. 421057 X 27358), 1µm Ø) to remove all particles. The clear aqueous filtrate was used as stock solution for preparation of the desired concentration range.

Test organisms (species):

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

Details on test organisms:

The test was carried out using the freshwater unicellular green algae Selenastrum capricornutum (new name: Raphidocelis subcapitata), purchased form Charles River Aquatic, Someren, The Netherlands). The algae were maintained on mineral medium and regularly transferred to a fresh mineral medium to act as inoculum. Reference tests with potassium dichromate will be performed every 6 months and compared with international inter-laboratory test data to confirm that the algae has not changed.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.2 - 23.1°C

pH:

7.7 - 8.2

Nominal and measured concentrations:

Nominal: 0.022, 0.044, 0.089, 0.178, 0.356 mg/L
Measured (geometric mean): -, -, 0.043, 0.051, 0.108 mg/L

Details on test conditions:

- Glassware: The test was performed in 300 mL Erlenmeyers containing 100 mL of mineral salts medium. The test flasks were closed with cotton-wool stoppers.
- Apparatus and conditions: The culturing cabinet was a temperature-controlled illuminated orbital incubator (IOI-400, Sanyo, Breda, The Netherlands) in which the temperature was maintained at 23 ± 2°C, and a continuous uniform illumination was provided in the spectral range of 400 to 700 nm by using 30 W fluorescent lamps of the type 'universal white' (colour temperature of approximately 4000 K), at a distance of 0.35 m from the algal cultures. The light intensity was in the range of 60 to 120 µmol (=µE) m2 s-1. The culture flasks were rotated continuously at approximately 100 rev/min to prevent sedimentation of the algae. The pH's were determined with the aid of a pH meter (pH 196, WTW, Weilheim, Germany). The temperature was measured continuously using a temperature sensor coupled to a datalogger (Elbanton, Kerkdriel, The Netherlands). The light intensity was measured with a light intensity meter (Li-Cor, Ll-189, Rijswijk, The Netherlands).
- Test inoculum: The initial stock culture was inoculated with Selenastrum capricornutum from a pre-culture and checked for purity by microscopic means (1000 times magnification) using an inverted microscope (Olympus IMT-2, Paes, Zoeterwoude, The Netherlands). Before the beginning of the test the extinction of an exponentially growing stock culture was measured. The cell density was determined using a calibration curve. From this algal culture a dilution was prepared to obtain an initial cell density of approximately 1E4 cells/mL in the medium of the test.
- Test medium: The test medium (mineral medium) was prepared and contained the following nutrients: Macro-nutrients (15 mg/L NH4Cl, 1.6 mg/L KH2PO4, 18 mg/L CaCl2(H2O)2, 15 mg/L MgSO4(H2O)7, 12 mg/L MgCl2(H2O)6, 0.08 mg/L FeCl3(H2O)6, 0.1 m/L Na2EDTA(H2O)2) and Trace elements (0.185 mg/L H3BO3, 0.003 mg/L ZnCl2, 0.340 mg/L MnCl2(H2O)2, 0.0015 mg/L CoCl2(H2O)6, 1E-5 mg/L CuCl2(H2O)2, 0.007 mg/L Na2MoO4(H2O)2, 50 mg/L NaHCO3). The pH of the medium was approximately 8. All solutions were made using deionized water with a conductivity of less than 0.5 µSmm-1 . The deionized water was prepared using a water purification system (Milli-Q, Milipore, Breda, The Netherlands).
- Determination of test parameters: Cell concentrations were determined photometrically, using a UV/VIS spectrophotometer (Shimadzu UV 210-PC, 's-Hertogenbosch, The Netherlands). Measurements were carried out at 685 nm in a cuvette with a light path of 10 cm (initially) after thoroughly mixing. To establish the relation between absorption and cell density, a calibration curve was made. Several dilutions were prepared from an exponentially growing algal culture. Of these diluted cultures the absorptions were measured at 685 nm using the above mentioned spectrophotometer. The cell density of the same samples (at least two) were determined using a microscope and a counting chamber. From the relation between absorption (A) and counted cell number (N in cells/µL) a calibration curve was calculated on the basis of the equation: N = 780.1 * A -8.433 (X2 = 0.00202). The calibration curve is checked for accuracy and linearity every 12 months. The pH of all test substance samples and controls were measured at the beginning (t=0h, in pooled samples for each concentration) and at the end of the test (t=72h, in all samples). During the test the temperature in the cabinet was monitored continuously and the light intensity was measured every 24 hours. At the end of the test 6 random samples (from each concentration one) were checked for purity and for possible microbial contamination by microscopical means at 1000 times magnification.
- Test methods: The test concentration range used in this study was derived from the results of preliminary investigations on the toxicity of the test substance to algae. In this investigation 100% growth inhibition was found at a concentration of 20% and a significant inhibition was still found at 0.8 % of the stock solution. The mineral medium (299 mL for each concentration) was prepared with filter sterilized (0.2 µm) deionized water, and subsequently inoculated (1 mL) with an exponentially growing culture of Selenastrum capricornutum to obtain a final density of 1E4 cells/mL. This medium was divided into three separate vessels. The test was performed using 3 replicates of each concentration and six replicates of a control culture, where no test substance was added. The absorption of all samples and controls were measured at 685 nm after approximately 0, 24, 48 and 72 hours of testing. Mineral medium was used as blanc in the spectrophotometer.
- Validity criteria: For the test to be valid, the cell density in the control cultures should have increased by a factor of at least 16 within 72 hours (OECD,EEC), The pH in the test medium should not deviate more than 1.5 pH unit (EEC). The lowest concentration tested should have no significant effect on the growth of the algae, and the highest concentration should inhibit growth by at least 50% relative to the control (OECD, EEC).
- Statistical evaluation: The values of the test substance were determined by comparing the relative growth (biomass) and growth rate (µ) of the algae at various concentrations of the test substance to the control cultures. Calculations were based on counted cell numbers (measured photometrically). The absorptions measured for each concentration were converted to cell numbers using the calibration curve. A correction for coloured samples was applied when necessary. Whenever the measured cell density at t=0 was below 1E4 cells/mL (due to extreme low extinction values at t=0) the density was corrected to this value for calculation. The ECn values for growth (EbC50) and growth rate (ErC50) were computed using a nonparametric (Spearman-Karber analysis) method (nominal concentrations) and the Probit analyses (measured concentrations). The values together with the 95% confidence limits were calculated using the computer program ToxCalc. Statistical comparisons of the growth and growth rate in control and test cultures for determination of the NOEC were carried out using the treshold values of the William's test. The computer program was validated using a known dataset before use. Calculation procedures are based on both nominal values (calculated from the measured concentration of the stock solution), and geometric means of measured values of the concentration range (whenever the recovery was < 80% after 72 hours of testing). The geometric means of concentrations which were not measured by chemical analysis, were estimated on the basis of the average values of the preceeding and following measured concentration at each sampling time. If values were below the detection limit, the detection limit values were used for calculation. Concentrations which were already below the detection limit at the start of the test, were not included in calculation procedures.

Reference substance (positive control):

yes

Remarks:

potassiumdichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.063 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.081 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.039 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.044 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Analysis results
- General: GC analysis of pure extraction solvent (dibutylether) and a test substance standard solution showed that the test substance appears as a single peak which is free from interferences from the solvent and solvent impurities. Analysed samples were test media samples containing high, medium, low and zero test substance dose concentrations. The samples were taken at the beginning of the test (t=0 hr), at the end of the test and between times after every 24 hours. After shaking and sonication, none of the samples contained visible particles or aggregates so none of the samples was centrifuged.
- Samples from algal growth inhibition test: The initial concentration of the test substance in the algal media was between 0 and 0.24 mg/L. The low dose concentration was already below the detection limit of 0.04 mg/L. During the 72 hour test the test substance concentrations were found to decrease steadily. At the end of the test concentration levels in the test media were all below the detection limit except for the highest dose concentration, which had declined to 0.05 mg/L. Extraction procedures for chemical analysis were performed using samples including algae, therefore the decrease could not be attributed to adsorption onto the algae. Moreover, adsorption onto the vessel wall is not likely to be the main cause of the decrease because the results of a photodegradation study on the test substance showed a maximum adsorption of 4.5% of the test substance after 72 hours of incubation.

Test environment
- The cell density of the algae in the control cultures increased with a factor 120 within 72 hours. The pH measurements show a maximum increase of 0.5 units. The temperature was measured continuously during the test and varied between 22.2 and 23.1°C, with an average value of 22.9°C. The light intensity was measured daily, and varied between 86.13 and 89.14 µmol m-2 s-1 during the test period. At the end of the test six samples were checked microscopically and all were pure and not contaminated with bacteria or other algae.

Test results
- Almost complete inhibition of both growth and growth rate of Selenastrum capricornutum was observed at 0.356 mg/L of the test substance, and no significant inhibition was observed at 0.044 mg/L test substance. There was however a substantial effect observed at the lowest concentration tested (0.022 mg/L). Due to this anomaly, the EC50 values based on the nominal values were calculated by the non-parametric Spearman-Karber method instead of the parametric Probit method, because the Spearman-Karber method provides fail safe EC50 values with 95% confidence limits and is resistant to anomalies as observed. The Trimmed Spearman-Karber method provides only EC50 values (with 95% confidence limits), and therefore the EC20 and EC80 could not be determined. Because the test concentrations dropped below 80% of the initial concentration, the calculation of the effects were also performed using the geometric means of the measured concentrations. For the calculation of the ECn values using the measured concentrations the Probit method was used (lowest concentrations (0.022 and 0.044 mg/L) were not included in calculation because the measured values were below the detection limit of the chemical analysis). The EbC50 derived from the results using the nominal values is 0.081 mg/L (0.069 - 0.095 95% confidence limits). The nominal ErC50 calculated from the results is 0.200 mg/L (0.179 - 0.221 95% confidence limits). The nominal NOEC determined using the treshold values of the Williams test is <0.022 mg/L for biomass and 0.044 mg/L based on growth rate. The anomaly observed at the lowest concentration was most apparent for the inhibition of growth and less evident for the inhibition of the growth rate. Therefore the NOEC based on the growth rate is probably more reliable. The EbC50 value based on the measured concentrations is 0.038 mg/L (0.032 - 0.042 95% confidence limits) and the ErC50 is 0.063 mg/L (0.059 - 0.069 95% confidence limits). The NOEC based on the measured concentrations was < 0.043 mg/L for biomass and 0.039 mg/L based on the growth rate. The calculation of both the ECn values and the NOEC's using the measured concentrations are based on limited data (only 3 values were used for calculation) and the data used were near the detection limit of the analysis, and therefore the results should be interpreted with care.

Validity of test
- The test was found valid by the increase of cell density of a factor of 120, a maximum pH deviation of 0.5 units and a inhibition of more than 50% at the highest concentration tested and no significant inhibition at the lowest concentration tested.

Results with reference substance (positive control):

The results of the last quality control test with potassiumdichromate (06-Feb-1998: EbC50, 0.51 mg/L - ErC50, 0.98 mg/L) are in agreement with the criteria mentioned in the EEC Directive 67/548 C3, indicating that the sensitivity of the algae has not changed significantly.

Validity criteria fulfilled:

yes

Description of key information

The 72h-ErC50 and 72h-NOErC are 0.063 and 0.039 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.063 mg/L

EC10 or NOEC for freshwater algae:

0.039 mg/L

Additional information

The toxicity to algae (Selenastrum capricornutum) of the test substance was determined in an acute toxicity test which is described in the OECD Guideline 201 and was performed in accordance with the principles of Good Laboratory Practice (GLP). Algae were exposed to the test substance at concentrations of 0.022, 0.044, 0.089, 0.178, and 0.356 mg/L for an exposure period of 72 hours. During the test the pH varied no more than 0.5 units, and the cell density increased with a factor of 120. The temperature varied between 22.2 and 23.1°C, and the light intensity between 86.13 and 89.14 µmol m-2 s-1. Almost complete inhibition of growth at the highest concentration and no significant inhibition of growth at the lowest concentration was observed during the test period, These data indicates that the validity criteria of the test was fulfilled. The EbC50 and ErC50 of the test substance based on the nominal values are 0.081 (0.069 - 0.095 95% confidence limits) and 0.200 (0.179 - 0221 95% confidence limits), respectively. The EbC50 and ErC50 of the test substance based on the measured values are 0.038 (0.032 - 0.042 95% confidence limits) and 0.063 (0.059 - 0.069 95% confidence limits), respectively. The NOEC based on the growth rate derived from the results is 0.044 mg/L and 0.039 mg/L for the nominal and measured concentrations, respectively. The calculation of the ECn values using the measured concentrations are based on limited data and the data used were near the detection limit of the analysis, and therefore the results should be interpreted with care. The chemical analyses indicated a rapid decrease of the concentration during the test period, which may be attributed to adsorption onto the test vessel wall.

In an additional test, the decrease in concentration during the acute algae and fish test was tried to be explained. It can be concluded that the test substance is not photo-degraded under the given test conditions of the OECD 201 Algae toxicity test. Furthermore, the decrease in concentration during the test cannot be attributed to adsorption to the test vessel wall as only 4.5 % of the total amount of test substance was extracted from the test vessel wall at the end of the test. However, a substantial fraction of the test compound is absorbed by the algae at the start of the test (approx 40%).