1,3-Benzodioxole-5-carboxylic acid, 3a,6,7,7a-tetrahydro-2,2-dimethyl-7-[(methylsulfonyl)oxy]-, ethyl ester, (3aR,7R,7aR)-

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-05-12 to 2006-06-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive and done to a valid guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl (3aR,7R,7aR)-2,2-dimethyl-7-[(methylsulfonyl)oxy]-3a,6,7,7a-tetrahydro-1,3-benzodioxole-5-carboxylate

EC Number:

606-548-2

Cas Number:

204254-84-2

Molecular formula:

C13 H20 O7 S

IUPAC Name:

Ethyl (3aR,7R,7aR)-2,2-dimethyl-7-[(methylsulfonyl)oxy]-3a,6,7,7a-tetrahydro-1,3-benzodioxole-5-carboxylate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

White to beige crystalline powder/solid.
Purity 99.2%
Expiry date: 21 December 2006

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Duplicate samples of each test medium and control were taken just before the start of the test and after 72 hours (all samples taken were without algae).All samples were deep-frozen (at about -20 °C) immediately after sampling. Based on pre-experiments (without GLP) for investigation of the storage stability, the test item is sufficiently stable in the test water under these storage conditions.
The concentration of the test item was analyzed in one of the duplicate test media samples from the test concentrations of nominal 10-100 mg/L from both sampling times (0 and 72 hours). The samples from the test concentrations of nominal 2.2 and 4.6 mg/L were not analyzed, since these concentrations were below the determined 72-hour NOEC. From the control samples, only one of the duplicate samples was analyzed from the corresponding sampling times.

Test solutions

Vehicle:

yes

Details on test solutions:

The test medium of the highest nominal test item concentration of 100 mg/L was prepared by dissolving 50.0 mg of the test item completely in 500 mL of test water using ultrasonic treatment (30 minutes) and intense stirring (30 minutes at room temperature in the dark). Adequate volumes of this intensively mixed test medium were diluted with test water to prepare the test media with the lower test item concentrations.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

The test organism used for the study was Scenedesmus subspicatus CHOOAT, Strain No. 86.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Piant Physiology, University of Göttingen, 0-37073 Göttingen, Germany). The algae are cultivated in RCC's laboratories under standardized conditions according to the test guidelines. The algae were cultivated and tested in synthetic test water, prepared according to the test guidelines. Analytical grade salts were dissolved in sterile purified water.

Study design

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Test conditions

Hardness:

water hardness of the test water: 0.24 mmol/L (= 24 mg/L as CaC03)

Test temperature:

23-24 °C

pH:

8.0 to 8.4 at start of test, 8.1 to 8.7 at the end of the test.

Nominal and measured concentrations:

The analytically determined test item concentrations in the analyzed test media of 10, 22, 46 and 100 mg/L from the start and the end of the test varied in the range of 78 to 94% of the nominal values. These are tabulated below. The samples from the test concentrations of nominal 2.2 and 4.6 mg/L were not analyzed, since these concentrations were below the determined 72-hour NOEC.

Details on test conditions:

The test was started (0 hours) by inoculation of 10,000 algal cells per mL of test medium. These cells were taken from an exponentially growing pre-culture that was set up three days prior to the test under the same conditions as in the test. The test design included three replicates per test concentration and six replicates of the control. Volumes of 15 mL algal suspension for each replicate were continuously stirred by magnetic stirrers in 50 mL Erlenmeyer flasks. The flasks were covered with glass dishes. They were incubated in a temperature controlled water bath at a temperature of 23-24 'C (Table 8), and continuously illuminated at a measured light intensity of about 9000 Lux (mean value), range: 8120 to 9550 Lux (minimum and maximum value of measurements at nine places distributed over the experimental area at the surface of the test media). This illumination was achieved by fluorescent tubes (Philips TLD 36W/840), installed about 35 cm above the test flasks. For evaluation of the algal quality and the experimental conditions, potassium dichromate is tested as a positive control at least once a year to demonstrate satisfactory conditions of the test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

23 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 11-53 (95%CL)

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

9.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 0.2-17 (95% CL)

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

54 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 31-n.d. (95% CL)

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 5

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

45 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 7

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

21 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 8

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

95 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 9

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 10

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The analytically determined test item concentrations in the analyzed test media of 10, 22, 46 and 100 mg/L from the start and the end of the test varied in the range of 78 to 94% of the nominal values. The mean measured concentrations (calculated as the average over all measurements per
test concentration) ranged from 85 to 90% of nominal. Consequently, the test item was sufficiently stable during the test period of 72 hours under the conditions of the test, and the reported biological results are based on the nominal concentrations of the test item.
The test item had a statistically significant inhibitory effect on the growth (biomass and growth rate) of Scenedesmus subspicatus after
the test period of 72 hours at concentrations of 22 mg/L and above. Thus, the test concentration of 22 mg/L was determined as the 72-hour LOEC (lowest concentration tested with toxic effects). The 72-hour NOEC (highest concentration tested without toxic effects after a test period of
72 hours) was determined to be 10 mg/L, since up to and including this test concentration both the mean biomass and the mean growth rate of the algae were not statistically significantly lower than in the control.
The microscopic examination of the algal cells after 72 hours exposure showed no difference between the algae growing in test medium with reduced algal growth (nominal 46 mg/L) and the algal cells in the control. There were no obvious effects on the shape and size of the algal cells growing in test media containing the test item at up to and including this nominal concentration.

Results with reference substance (positive control):

potassium dichromate is tested as a positive control at least once a year to demonstrate satisfactory conditions of the test

Reported statistics and error estimates:

The EC50 values (the respective concentrations of the test item corresponding to 50% inhibition of algal biomass (b) and growth rate (r) compared to the control), and the corresponding ECl0 value and EC90 values and their 95%confidence limits were calculated by Probit Analysis.
For the determination of the LOEC and NOEC, the calculated mean biomass and the mean growth rate at the test concentrations were tested for significant differences when compared to the control values by a Dunnett-test.

Any other information on results incl. tables

In the control the cell density increased from a nominal N = 1 x 10⁴ cells/mL at the start of the test (0 hours) to N = 95 x 10⁴ cells/mL (mean value) after 72 hours. Thus the algal growth in the control was sufficiently high under the conditions of the test. The validity criterion of increase of cell density by at least a factor of 16 over 72 hours was fulfilled. No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The test item had a statistically significant inhibitory effect on the growth (biomass and growth rate) of Scenedesmus subspicatus after the test period of 72 hours at concentrations of 22 mg/L (= 72-hour LOEC: lowest concentration tested with toxic effects) and above. The 72-hour NOEC (highest concentration tested without toxic effects after a test period of 72 hours) was determined to be 10 mg/L, since up to and including this test concentration both the mean biomass and the mean growth rate of the algae were not statistically significantly lower than in the control.

Executive summary:

The influence of the test item on the growth of the green algal species Scenedesmus subspicatus was investigated in a 72-hour static test. The nominal test concentrations were 2.2, 4.6, 10, 22, 46 and 100 mg/L in parallel with a control.

The test item had a statistically significant inhibitory effect on the growth (biomass and growth rate) of Scenedesmus subspicatus after the test period of 72 hours at concentrations of 22 mg/L (= 72-hour LOEC: lowest concentration tested with toxic effects) and above. The 72-hour NOEC (highest concentration tested without toxic effects after a test period of 72 hours) was determined to be 10 mg/L, since up to and including this test concentration both the mean biomass and the mean growth rate of the algae were not statistically significantly lower than in the control.

The EC values as well ás the NOEC and LOEC were calculated for both parameters, the algal biomass (b) and the growth rate (r), after 72 hours test duration:

Parameter (0 -72 h)	Biomass b (mg/L)	Growth rate r (mg/L)
EC50 (95%-confidence limits)	23 (11 -53)	45 (n.d.)
EC10 (95%-confidence limits)	9.8 (0.2 -17)	21 (n.d.)
EC90 (95%-confidence limits)	54 (31 -n.d.)	95 (n.d.)
NOEC	10	10
LOEC	22	22

n.d. - could not be determined