Dicyclopentyldimethoxysilane

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:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

92/69/EEC

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all concentrations

- Sampling method: the stability of the test solutions without organisms was analysed at all concentrations at 0h and 72 h. The test medium in the study were taken from the solvent control and each test group (replicates R1-R3) at 0 and 72 hours for quantitative analysis.

- Sample storage conditions before analysis: not reported.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

- Method: amounts of test material (0.8, 1.4, 2.5 and 4.5 g) were dispersed in auxiliary solvent and the volume adjusted to 10 ml to give stock solutions of 0.8, 1.4 2.5 and 4.5 g/10ml. A dilution was made from the 4.5 g/10 ml stock solution to produce a 0.45g/10 ml stock solution. Aliquots (100 ul) of each stock solution were dispersed in 1 L of algal suspension to give the test concentrations of 4.5, 8.0, 14, 25 and 45 mg/l.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): 7% v/v Tween 80-Acetone

- Concentration of vehicle in test medium: 10ul

- Evidence of undissolved material (e.g. precipitate, surface film, etc): none reported

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM

- Strain: CCAP 276/20

- Source (laboratory, culture collection): liquid cultures were obtained from CCAP, Institute of Freshwater Ecology, Ferry House, Ambleside, Cumbria, UK. Cultures were maintained in house.

- Age of inoculum (at test initiation): 1 week

- Method of cultivation: from the original cultures the culture medium was replenished once per week


ACCLIMATION

- Acclimation period: cultures maintained in house

- Culturing media and conditions (same as test or not): conditions similar to test: 21degC, continuous illumination (7000 lux) and constant aeration.

- Any deformed or abnormal cells observed: none reported

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24 degrees C throughout the study

pH:

from 7.9-8.0 at 0 h to 9.9-10.1 at 72 h

The increase of pH is caused by the algal growth in the test vessels. In common with other species of freshwater algae, Desmodesmus subspicatus gains CO2 from HCO3-, leading to a release of hydroxide ions, that alkalise the medium.

The shift in pH of >1 unit is indicative of good algal growth and is not considered to invalidate the test result.

Nominal and measured concentrations:

Nominal concentrations: 0, 4.5, 8.0, 14, 25 and 45 mg/L.

Details on test conditions:

TEST SYSTEM

- Test vessel: conical flasks

- Material, size, headspace, fill volume: 250 ml falsks containing 100 ml solution.

- Aeration: none reported

- Initial cells density: 1.23 x 10^4 cells/ml, solvent control 9.17 x 10^3 cells/ml

- Control end cells density: 6.8 x 10^5 cells/ml, solvent control 5.98 x 10^5 cells/ml

- No. of vessels per concentration (replicates): 3

- No. of vessels per control (replicates): 3

- No. of vessels per vehicle control (replicates): 3


GROWTH MEDIUM

- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS

- Source/preparation of dilution water: not reported

- Culture medium different from test medium: no

- Intervals of water quality measurement: 24 h


OTHER TEST CONDITIONS

- Sterile test conditions: not reported

- Adjustment of pH: none reported

- Photoperiod: continuous

- Light intensity and quality: 7000 lux


EFFECT PARAMETERS MEASURED:

- Determination of cell concentrations: counting chamberl; samples taken at 0, 24, 48 and 72 h.


TEST CONCENTRATIONS

- Spacing factor for test concentrations: 1.8

- Range finding study

- Test concentrations: 10 and 45 mg/l

- Results used to determine the conditions for the definitive study: NOEC 10 mg/l; growth observed to be reduced at 45 mg/l.

Reference substance (positive control):

no

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

5.8 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

organisms also exposed to hydrolysis product

Basis for effect:

growth rate

Remarks on result:

other: equivalent to 10.3 mg/l geometric mean

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(organisms also exposed to hydrolysis product)

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.94 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate and biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(organisms also exposed to hydrolysis product)

Basis for effect:

other: growth rate and biomass

Remarks on result:

other: equivalent to 3.6 mg/l geometric mean

Details on results:

- Exponential growth in the control (for algal test): yes

- Aggregation of algal cells: at 14 mg/l cells were clumped

- Other: at 25 and 45 mg/l the cells were smaller in size than those in the control cultures.

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none reported

- Effect concentrations exceeding solubility of substance in test medium: yes, the water solubility of the test substance is 5.32 mg/l. However the water solubility of the hydrolysis product is 2600 mg/l

Reported statistics and error estimates:

One way analysis of variance

Table 1. Analytical monitoring results at 0 h and 72 h.

Nominal concentration	0 h		72h
	concentration found (mg/l)	% nominal	concentration found (mg/l)	% nominal
Control	<LOQ	-	<LOQ	-
4.5	0.519	12	0.0562	1
8.0	6.76	85	1.94	24
14.0	13.1	94	5.28	38
25.0	23.5	94	7.99	32
45.0	40.1	89	11.3	25

The corresponding geometric means have been calculated by the reviewer on the basis of the above data:

Nominal concentration (mg/l)	Geometric mean (mg/l)
Control	<LOQ
4.5	0.17
8	3.6
14	8.3
25	13.7
45	21.3

The authors of the report have written:

"Analysis of the solvent stock solutions used to prepare the test series at 0 h showed the measured concentrations to be near nominal. Analysis of the test solutions at 0 h showed the measured concentrations to be in excess of the required 80% of nominal with the exception of the 4.5 mg/l test group which was 12% of nominal. The low measured concentrations for the 4.5 mg/l test group at 0 h is considered to be due to sampling/analytical variation given that all other measured concentrations were in excess of 80% at 0 hours.

Analysis of the 72 h showed a marked decline in measured concentrations ranging from 1 to 25% of nominal.

Given that the pre-study stability analysis performed showed the test material to be stable over the study period, the decline in measured test concentrations is considered to be due to adsorption to the glassware and/or algal cells present.

The pre-study stability analysis did not reveal whether the test material adsorbed to glassware. This is considered to be due to the test vessels used in the stability analysis being rinsed with extraction solvent prior to analysis. This procedure was not performed on the test vessels used in the main study and may account for the decline in measured test concentrations after 72 h."

It is of the reviewers opinion that the report does not give sufficient information on the medium in which the stability test was conducted, i.e. it is unclear whether a solvent or the test medium was used. If the stability study was conducted within the solvent matrix this would explain the different results in test substance concentration in the stability study and in the definitive study. Since the 0 h concentration results in the definitive study are close to nominal, and the substance is known to hydrolyse (explaining the loss of test substance at 72 h), the definitive study concentration results are more in line with expectations. Therefore the results are based on nominal concentrations.

Table 2. Biological findings.

Nominal concentrations (mg/l)	Mean cell densities		Growth rate		Area under the curve
	0 h	72 h	(0-24 h)	% inhibition	72 h
Control	1.23 x 10^4	6.8 x 10^5	0.080	-	1.56 x 10^7	-
Solvent control	9.17 x 10^3	5.98 x 10^5	0.095	-	1.50 x 10^7	-
4.5	9.17 x 10^3	5.83 x 10^5	0.096	-	1.49 x 10^7	1
8.0	9.17 x 10^3	6.11 x 10^5	0.096	-	1.45 x 10^7	3
14	1.55 x 10^4	4.31 x 10^5	0.065	32	1.17 x 10^7	22
25	9.17 x 10^3	0.17 x 10^3	0.012	87	1.51 x 10^5	99
45	9.17 x 10^3	6.11 x 10^3	0.000	100	-3.67 x 10^4	100

Mean cell densities from 3 replicates.

Validity criteria fulfilled:

yes

Conclusions:

A 0-24h ErC50 nominal value of 18 mg/l, equivalent to 5.8 mg/l measured (arithmetic mean), has been determined for the effects of the test substance on the growth rate of Scenedesmus subspicatus (new name: Desmodesmus subspicatus). A 72 h NOEC nominal value of 8 mg/L, equivalent to 1.9 mg/l measured (arithmetic mean), has also been determined for the effects on growth and biomass. Test organisms are likely to have been exposed to a mixture of the parent substance and the hydrolysis product.

Description of key information

(0-24 h) ErC₅₀ 10 mg/l (geometric mean), reliability 1, D. subspicatus (parent substance)

(72 h) NOEC 3.6 mg/l (geometric mean), reliability 1, D. subspicatus (parent substance)

Key value for chemical safety assessment

EC50 for freshwater algae:

10 mg/L

EC10 or NOEC for freshwater algae:

3.6 mg/L

Additional information

A 0-24 -hour E_rC₅₀ value nominal of 18 mg/l (corresponding to a parent substance concentration of 10 mg/l, geometric mean) has been determined for the effects of the test substance on the growth rate of Desmodesmus subspicatus (tested as: Scenedesmus subspicatus). A 72-hour NOEC value of 8 mg/l (corresponding to a parent substance concentration of 3.6 mg/l, geometric mean) has also been determined for the effects on growth and biomass. Test organisms are likely to have been exposed to a mixture of the parent substance and the hydrolysis product. This study (Safepharm Laboratories Ltd. 1996d) has been selected as key since it is the lowest reliable value available with the test substance.

In the Safepharm Laboratories Ltd. 1996d study GC analysis was conducted to measure the concentration of dicyclopentyl(dimethoxy)silane in the exposure test media. The analysis indicates that the half-life of the test material in the test media during this study was approximately 24 hours, at 24⁰C and pH range 8-10.

Another reliable value has determined an EC₅₀ of 20 mg/l. Two studies of reliability 4 reported EC₅₀ values of 0.36 and 4.8 mg/l.

The reliability of the Corning Hazleton (1996c), which determined an EC50 value of 0.36 mg/l, could not be assigned because it is not possible to ascertain whether nominal and measured concentrations in the test media were in good agreement. The stock solution was prepared over a period of 5 hours; during this time the test substance will have partially hydrolysed before serial dilutions took place. Additionally, chemical analysis was not successful in aqueous solutions. However, the dose-response curve of the study does not appear to be anomalous. Therefore, while the Corning Hazelton (1996b) has been assigned reliability 4 due to potential for nominal and exposure concentrations being poorly correlated and the study has not been used for the derivation of PNECs, the study does provide evidence that the registered substance is toxic to algae and the study results have been used for the purposes of classification and labelling.

One other available result has been extracted from a summary report to which reliability could also not be assigned due to lack of information; the report presents the same result presented in La Noyearie (1989).