Chlorotrioctylstannane

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:

May 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

see below

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

see below

Principles of method if other than guideline:

The test employs only 4 different concentrations, rather than the 5 specified in the guidelines. As well as three dilutions of the test material in water, an additional test solution was prepared with a nominal loading of 1.2 mg/L using a vehicle of Tween 80. This is not considered to have any impact on the validity or reliability of the study.

GLP compliance:

yes

Specific details on test material used for the study:

- Storage conditions of test material: ~ 4 ° C, in the dark in dry conditions
- Expiry date: at least May 99

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Each test solution, including the controls.
- Sampling method: Samples for analytical measurements were taken directly after preparation.
- Sample storage conditions before analysis: Deep frozen at -18 °C until analysis.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For the stock solution (without vehicle) 130 mg of test material was dispersed in 1000 mL of demineralised water and stirred for approximately 24 hours (pH 6.2). Aliquots of this unfiltered Water Accommodated Fraction (WAF )were diluted 1:10 and 1:100 with demineralised water in order to prepare the appropriate test concentrations.
For the stock solution including the vehicle, 1.2 mg test material was dissolved in 102 mg/L Tween 80. This solution was furthermore dissolved in 1000 mL demineralised water (pH 8.5).
The vehicle control was prepared with 104 mg Tween 80 in 1000 mL demineralised water (pH 9.0).

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Strain: Scenedesmus subspicatus
- Method of cultivation: Stored on slant agar containing a nutrient solution at 10 to 15 °C under diffuse light; the culture was dissolved in a liquid nutrient solution and subsequently transferred approximately once weekly.

ACCLIMATION
- Acclimation period: The algae for the pre-culture were taken from a stock up to seven days old, approximately 72 hours before the start of the incubation in the test.
They were kept in concentrated nutrient solution (diluted 1:10) starting with an approximate cell concentration of 10 000 cells/mL, counted with the Coulter Counter.
This pre-culture was incubated for 3 days in an incubator shaker apparatus. The temperature was held at approximately 24 °C. After the incubation period the cell concentration of the algae was measured (~ 1 172 400 cells/mL), and the pre-culture was diluted to 10 000 cells per mL with demineralised water, ready to be used as inoculum.
- Culturing media and conditions (same as test or not): Yes

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24 °C

pH:

8.2 to 9.1

Nominal and measured concentrations:

- Nominal concentrations: 130 mg/L saturated WAF and 1:10 and 1:100 dilutions of the WAF. Additionally, 1.2 mg/L test material in a solution with 100 mg/L Tween 80.
- Measured concentrations: The concentration of the saturated WAF was <0.07 mg/L. The concentration of the solution prepared with Tween 80 was 0.4 mg/L.

Details on test conditions:

TEST SYSTEM
- Test solutions: 10 mL of the concentrated nutrient solution was added to 80 mL of test material solutions. Finally, 10 mL of the inoculum was added.
- Aeration: The algae were incubated in an incubator shaker
- Initial cell density: Approximately 10 000 cells/mL
- Control end cell density: 7.16 x 105 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 3

GROWTH MEDIUM
- Standard medium used: Yes
Preparation of nutrient solution for pre-culture and test inoculum: 100 mL of solution 1, 10 mL of solution 2, and 0.1 mL of solution 3 were added and filled up to 1000 mL with demineralised water, stirred under aeration for ca. 1 hour and filtered under sterile conditions.
- Solution 1: 160 mg potassium dihydrogenphosphate, 8 mg ferrous (III) chloride x 6H2O, 10 mg titriplex III x 2H2O, 19 mg boric acid and 42 mg manganese chloride x 4H2O were dissolved in 1000 mL demineralised water.
- Solution 2: 30 mg zinc chloride, 15 mg cobalt chloride x 6 H2O and 70 mg sodium molybdate x 2 H2O were dissolved in 1000 mL demineralised water and thereafter diluted 1:10.
- Solution 3: 10 mg copper chloride x 2 H2O was dissolved in 100 mL demineralised water and thereafter diluted 1:100.
- Concentrated nutrient solution: 150 mg ammonium chloride, 120 mg magnesium chloride x 6 H2O, 180 mg calcium chloride x 2 H2O, 150 mg magnesium sulphate x 7 H2O and 500 mg sodium hydrogencarbonate were dissolved in around 500 mL of demineralised water.

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: No
- Intervals of water quality measurement: pH: was measured in all test vessels at the beginning and at the end of the test.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No

EFFECT PARAMETERS MEASURED (with observation intervals if applicable)
- Determination of cell concentrations: Cell concentrations of test and control solutions were measured after 24, 48 and 72 hours of incubation. All measurements were made on an electronic particle counting apparatus (Coulter Counter), which was calibrated twice a year and checked prior to taking the measurements.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 130 other: mg/L saturated WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The test material seemed to produce an inhibitory effect both on biomass and growth rate at the highest concentration (saturated solution). However, due to the relatively large coefficient of variation of algae cells in the control and the saturated solution the difference in growth is not likely to be of considerable extent. Furthermore, the inhibition would have been below 50 % and, therefore, an EC50 was not calculated.
The vehicle Tween 80 apparently produced an inhibitory effect on the cell growth (approximately 50 % of inhibition in the vehicle control compared to the control group). Therefore, the test concentration with addition of Tween 80 was excluded from evaluation.
The test material had a slight inhibitory effect on the growth of Scenedesmus subspicatus up to the solubility limit of 0.1 mg/L. The EC50 was higher than a saturated WAF.

ANALYSIS OF CONCENTRATION
The analysis of the saturated WAF gave varying results over 3 replicate analyses. One explanation may be the inhomogeneity of the test material in aqueous dispersions, which yielded different results. It has also to be noted that the extraction volume differed from 3 to 10 mL, which may also account for some variation in the analytical result. The solution prepared by means of a vehicle showed no relevant variation between the replicates.

Table 1: Numbers of algae cells (mean of n-replicates) at 24, 48 and 72 hours

Test Solution	Cell numbers (cells/mL)
	24 h	48 h	72 h
Control (n = 6)	3.4 x 10⁴	1.62 x 10⁵	7.16 x 10⁵
1:100 (n = 3)	3.9 x 10⁴	1.87 x 10⁵	8.45 x 10⁵
1:10 (n = 3)	3.4 x 10⁴	1.66 x 10⁵	6.16 x 10⁵
Saturated WAF (n = 3)	4.8 x 10⁴	1.36 x 10⁵	3.81 x 10⁵
0.0 mg/100 mg Tween 80 (n = 3)	4.3 x 10⁴	1.80 x 10⁵	3.61 x 10⁵
1.2 mg/100 mg Tween 80 (n = 3)	4.5 x 10⁴	2.39 x 10⁵	4.28 x 10⁵

 

Table 2: Biomass, growth rate and inhibition of biomass and growth rate after 72 hours

Test Solution	Area below the growth curves (x 10⁷ x h)	Percentage inhibition of the growth (biomass) IAi	Average specific growth rates (h^-1)	Percentage inhibition of the growth rate Iµi
Control (n = 6)	1.2697	0	0.0593	0
1:100 (n = 3)	1.4956	-17.8	0.0613	-3.4
1:10 (n = 3)	1.1595	8.7	0.0572	3.5
Saturated WAF (n = 3)	0.8379	34.0	0.0501	15.5
0 mg/100 mg Tween 80 (n = 3)	Not calculated - vehicle produced inhibitory effects on the cell growth
1.2 mg/100 mg Tween 80 (n = 3)

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, it was determined the test material had a slight inhibitory effect on the growth of Scenedesmus subspicatus up to the solubility limit of 0.1 mg/L. The EC50 was higher than a saturated WAF.

Executive summary:

The toxicity of the test material to the freshwater alga, Scenedesmus subspicatus, was investigated in a study which was conducted in accordance with the standardised guidelines OECD 201 and EU Method C.3 under GLP conditions.

The test material was incubated in an aqueous solution including nutrients with an algae population of Scenedesmus subspicatus for approximately 72 hours at 24 °C. For preparation of the test solutions, a saturated water accommodated fraction (WAF) was prepared by dispersing 130 mg of test material per litre of demineralised water. This WAF was used as the highest test concentration and as the basis for further dilutions of 1:10 and 1:100 with demineralised water. Additionally, a further test solution was evaluated that contained 1.2 mg/L test material with 0.1 g of Tween 80. Blank and solvent controls were tested concurrently.

Samples of the stock solution (prepared from the saturated WAF) and the solution with Tween 80 were taken directly after preparation for a concentration analysis by GC/FPD. The concentration of the saturated WAF was determined to be < 0.07 mg/L. The concentration of the solution prepared with Tween 80 was determined to be 0.4 mg/L.

As a parameter for the growth of the algae population, the cell concentrations of the test and control solutions were counted with an electronic particle counter and the inhibition of biomass and the growth rate were calculated on the basis of the cell counts. The calculated biomass and growth rate of each concentration were compared to those of the controls, and the inhibition was calculated.

The test material seemed to produce an inhibitory effect both on biomass and growth rate at the highest concentration (saturated solution). However, due to the relatively large coefficient of variation of algae cells in the control and the saturated solution the difference in growth is not likely to be of considerable extent. Furthermore, the inhibition would have been below 50 % and, therefore, an EC50 was not calculated.

The vehicle Tween 80 apparently produced an inhibitory effect on the cell growth (approximately 50 % of inhibition in the vehicle control compared to the control group). Therefore, the test concentration with addition of Tween 80 was excluded from evaluation.

The test material had a slight inhibitory effect on the growth of Scenedesmus subspicatus up to the solubility limit of 0.1 mg/L. The EC50 was higher than a saturated WAF.

The vehicle Tween 80 apparently produced an inhibitory effect on the cell growth (approximately 50 % of inhibition in the vehicle control compared to the control group). Therefore, the test concentration with addition of Tween 80 was excluded from evaluation.

Under the conditions of this study, it was determined the test material had a slight inhibitory effect on the growth of Scenedesmus subspicatus up to the solubility limit of 0.1 mg/L. The EC50 was higher than a saturated WAF.

Description of key information

72 hr EC50 >saturated WAF, Scenedesmus subspicatus, OECD 201 and EU Method C.3, Länge & Wendt (2000)

Additional information

The toxicity of the test material to the freshwater alga, Scenedesmus subspicatus, was investigated in a study which was conducted in accordance with the standardised guidelines OECD 201 and EU Method C.3 under GLP conditions. The study was assigned a reliability score of 2 in accordance with the principles for assessing data quality set forth by Klimisch et al. (1997).

The test material was incubated in an aqueous solution including nutrients with an algae population of Scenedesmus subspicatus for approximately 72 hours at 24 °C. For preparation of the test solutions, a saturated water accommodated fraction (WAF) was prepared by dispersing 130 mg of test material per litre of demineralised water. This WAF was used as the highest test concentration and as the basis for further dilutions of 1:10 and 1:100 with demineralised water. Additionally, a further test solution was evaluated that contained 1.2 mg/L test material with 0.1 g of Tween 80. Blank and solvent controls were tested concurrently.

Samples of the stock solution (prepared from the saturated WAF) and the solution with Tween 80 were taken directly after preparation for a concentration analysis by GC/FPD. The concentration of the saturated WAF was determined to be < 0.07 mg/L. The concentration of the solution prepared with Tween 80 was determined to be 0.4 mg/L.

As a parameter for the growth of the algae population, the cell concentrations of the test and control solutions were counted with an electronic particle counter and the inhibition of biomass and the growth rate were calculated on the basis of the cell counts. The calculated biomass and growth rate of each concentration were compared to those of the controls, and the inhibition was calculated.

The test material seemed to produce an inhibitory effect both on biomass and growth rate at the highest concentration (saturated solution). However, due to the relatively large coefficient of variation of algae cells in the control and the saturated solution the difference in growth is not likely to be of considerable extent. Furthermore, the inhibition would have been below 50 % and, therefore, an EC50 was not calculated.

The vehicle Tween 80 apparently produced an inhibitory effect on the cell growth (approximately 50 % of inhibition in the vehicle control compared to the control group). Therefore, the test concentration with addition of Tween 80 was excluded from evaluation.

The test material had a slight inhibitory effect on the growth of Scenedesmus subspicatus up to the solubility limit of 0.1 mg/L. The EC50 was higher than a saturated WAF.

The vehicle Tween 80 apparently produced an inhibitory effect on the cell growth (approximately 50 % of inhibition in the vehicle control compared to the control group). Therefore, the test concentration with addition of Tween 80 was excluded from evaluation.

Under the conditions of this study, it was determined the test material had a slight inhibitory effect on the growth of Scenedesmus subspicatus up to the solubility limit of 0.1 mg/L. The EC50 was higher than a saturated WAF.