Tin tetrachloride

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:

2009-11-09 to 2010-03-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study followed OECD Guidance under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ISO 14442 Water Quality - Guidelines for Algal Growth Inhibition Tests with Poorly Soluble Materials,Volatile Compounds, Metals and Waste Water, dated 2006

Principles of method if other than guideline:

This study was designed to comply with the following methods:
- OECD Guideline for Testing of Chemicals, Section 2, No. 201: "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", adopted March 23, 2006
- Commission Directive 92/69/EEC, Annex Part C, C.3: "Algal Inhibition Test", Official Journal of the European Communities No. L 383 A, dated December 29, 1992
- International Organisation for Standardisation. ISO 14442 Water Quality - Guidelines for Algal Growth Inhibition Tests with Poorly Soluble Materials,Volatile Compounds, Metals and Waste Water, dated 2006
- OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures", December 15, 2000

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Tin tetrachloride

EC Number:

231-588-9

EC Name:

Tin tetrachloride

Cas Number:

7646-78-8

Molecular formula:

Cl4Sn

IUPAC Name:

tin tetrachloride

Details on test material:

- Name of test material (as cited in study report): Tin tetrachloride
- Physical state: Liquid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

In order to prevent possible precipitation of tin the pH of the samples was adjusted to a value of 2 using hydrochloric acid directly after sampling. These samples were stored in a refrigerator (4 ± 4 °C), protected from light until analysis was performed. Before measurement the biological and fortified samples were ultrasonicated for 5 min.
Sampling: Duplicate samples from the freshly prepared and filtrated test media (containing algae) of all test concentrations and from the control were taken at the start of the test. For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test media of all test concentrations and the control (containing algae) were taken at the end of the test (after the 72 hours test period).

Test solutions

Vehicle:

no

Details on test solutions:

The test item or rather its hydrolysis products were not well soluble in test water. In this study, only the inhibitory effects of dissolved test item were tested and thus no concentrations above the solubility limit of the test item in test water were tested. Therefore suspensions with different loading rates were prepared from the test item by mixing adequate volumes of the test item into test water (exact data were documented in the raw data) and the pH of every test solution was adjusted to 8.1. These suspensions were carefully stirred for about 24 hours in the dark to reach the solubility equilibrium. Then, non-dissolved fractions of the test item were separated from the medium by membrane filtration (0.45 µm Cellulose Nitrate filter). The filtrates of the suspensions were used as test medium. Additionally, a control was tested in parallel.
The test media were prepared just before introduction of the algae (= start of the test).

Test organisms

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata, Strain No. 61.81 SAG
Origin: The algae were supplied by the "Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen", 37073 Göttingen, Germany.
Breeding Conditions: The algae were cultivated in the laboratories of IBACON under standardised conditions according to the test guidelines.
Reference Item: For the evaluation of the quality of the algae and the experimental conditions the reference item potassium dichromate p.a. was tested to demonstrate satisfactory test conditions.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Nominal and measured concentrations:

Nominal (Loading rate): 100, 32, 10, 3.2, 1 mg/L

Details on test conditions:

TEST UNITS
Type and Size: Erlenmeyer flasks of 50 mL volume with 50 mL of test medium.
Identification: Each test unit was uniquely identified with the serial number of the study, treatment and replicate number.

TEST CONDITIONS
Water Temperature: The temperature was measured daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks.
Water temperature was: 23 - 24 °C (see Table 9)
pH-Values: The pH was measured in all test item concentrations and the control at the start and the end of the test.
pH was: 8.1 at test start and 8.1 at test end (see Table 8)
Light Regime: Continuous illumination
Light Intensity: The light intensity was measured once during the test at 6 positions distributed over the experimental area at the surface of the test media.
Mean light intensity: 8573 lux (range: 8470-8700 lux)
Recording: Test conditions were recorded with suitable instruments and documented in the raw data.

COURSE OF THE TEST
Introduction of Algae: The test was started (0 hours) by inoculation of a biomass of 10000 algal cells per mL test medium. These cells were taken from an exponentially growing pre-culture, which was set up 4 days prior to the test start under the same conditions as in the test.
Replicates: The test was performed with three replicates per test concentration and six replicates in the control. Volumes of 50 mL of algal suspension per replicate were continuously stirred with magnetic stirrers in 50 mL Erlenmeyer flasks. The flasks were covered with glass dishes and incubated in a water bath.
Blanks: Additionally, one replicate of each test concentration and of the control was prepared without algae to provide a "blank" for the spectrophotometrical measurements. The additional replicates were incubated under the same conditions as described above. The blank values were subtracted from the absorption measured in the samples containing algae in order to eliminate absorption caused by test item (see also "Determination of the Cell Density").


TEST MEDIUM
Analytical grade salts were added at the following nominal concentrations in deionised water (conductivity <5 µScm-1):
Macro-nutrients: NaHCO3 50.0 mg/L
CaCl2  2 H2O 18.0 mg/L
NH4Cl 15.0 mg/L
MgSO4  7 H2O 15.0 mg/L
MgCl2  6 H2O 12.0 mg/L
KH2PO4 1.6 mg/L
Trace Elements: C6H5O7Fe  H2O 64.0 µg/L
C6H8O7  H2O 6.0 µg/L
MnCl2  4 H2O 415.0 µg/L
H3BO3 185.0 µg/L
Na2MoO4  2 H2O 7.0 µg/L
ZnCl2 3.0 µg/L
CoCl2  6 H2O 1.5 µg/L
CuCl2  2 H2O 0.01 µg/L
Buffer: HEPES 2.98 g/L (2-(4-(2-Hydroxylethyl)-1-piperazinyl)-Ethansulfonsäure

Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L) as CaCO3.
In all stock solutions the pH was adjusted with 1 M NaOH to pH 8.1.

Reference substance (positive control):

yes

Remarks:

potassium dichromate in a separate experimental study ( IBACON Project Nr. 52081210)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Loading rate

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

< 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Loading rate

Basis for effect:

growth rate

Details on results:

BIOLOGICAL RESULTS
Cell Density Increase in Control Cultures: 95.7-fold increase within 72 hours and thus, validity criterion was met.
Coefficient of Variation of Sectional (Daily) Growth Rates in Control Cultures: 20.9 % and thus, validity criterion was met.
Coefficient of Variation of Average Growth between Control Replicates: 1.0 % and thus, validity criterion was met.
Growth Inhibition: The 72-hour ErL50 value was observed to be >100 mg test item/L, the 72-hour EbL50 was determined to be between 1.0 and 3.2 mg test item/L and the 72-hour EyL50 was observed to be < 1.0 mg test item/L. The 72-hour NOELR were determined to be < 1.0 and the associated 72-hour LOELR are ≤ 1.0 mg/L for all endpoints. Statistical analysis to get the true EL50 values was not practicable due to the absence of a dose response relationship above 3.2 mg test item/L. This is in the line with saturation effects. Below the concentration of 3.2 mg/L the inhibition was distinctly lower, indicating that a proper dose range was selected for the experiment.

ANALYTICAL RESULTS
Determination of the Test Item: Based on the results of AAS measurements the concentration of the tin was determined using a calibration curve.
Calibration Range: 0.005 to 0.1 mg/L
Linearity of Response: Correlation of peak area of different standard solutions with their corresponding concentrations, using a linear regression
Regression Coefficient: 0.9987
Calibration Curve: y = 0.8422 * x + 3.6631 (see also Figure 2)
Limit of Detection: 0.006 mg Sn/L
Limit of Quantification: 0.01 mg Sn/L
Mean Recovery in the Fortified Samples: 90 % (n = 11, RSD 10 %)
Mean Recovery in the Test Samples: All values are below the limit of detection of the analytical method
The quantification of the test item in the test media was not possible. Due to the low water solubility of the test item the concentrations of test item in the test media were below the limit of detection of the analytical method which is determined to be 0.006 mg Sn/L.

Results with reference substance (positive control):

- EC50 72h Growth rate: 0.45 mg/L

Reported statistics and error estimates:

Statistical analysis to get the true EL50 values was not practicable due to the absence of a dose response relationship above 3.2 mg test item/L. This is in the line with saturation effects.

Any other information on results incl. tables

Table 1. Biological results

Parameter (0 - 72 h)	Growth rate [mg test item/L]	Biomass [mg test item/L]	Yield [mg test item/L]
72-hour EL50	> 100	> 1.0< 3.2	< 1.0
95 % conf. limits	n.d.	n.d.	n.d.
72-hour EL10	< 1.0	< 1.0	< 1.0
95 % conf. limits	n.d.	n.d.	n.d.
72-hour NOELR	< 1.0	< 1.0	< 1.0
72-hour LOELR	≤ 1.0	≤ 1.0	≤ 1.0
n.d. = not determinable
Values refer to nominal test concentrations (loading rates)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The influence of Tin Tetrachloride on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static dose-response test. The 72-hour ErL50 value was observed to be >100 mg test item/L, the 72-hour EbL50 was determined to be between 1.0 and 3.2 mg test item/L and the 72-hour EyL50 was observed to be < 1.0 mg test item/L. The 72-hour NOELR were determined to be < 1.0 and the associated 72-hour LOELR are ≤ 1.0 mg/L for all endpoints.

Executive summary:

Tin Tetrachloride was examined for toxicity on freshwater green algae Pseudokirchneriella subcapitata in a static dose-response test according to OECD Guideline 201 and ISO 14442 Water Quality - Guidelines for Algal Growth Inhibition Tests with Poorly Soluble Materials,Volatile Compounds, Metals and Waste Water. The medium was EDTA free to prevent from complexation of free tin(IV). The validity of the test desing was evaluated in a separate study with poatassium dicromate. All validity criteria were fullfilld. The 72-hour ErL50 value was observed to be >100 mg test item/L, the 72-hour EbL50 was determined to be between 1.0 and 3.2 mg test item/L and the 72-hour EyL50 was observed to be < 1.0 mg test item/L. The 72-hour NOELR were determined to be < 1.0 and the associated 72-hour LOELR are ≤ 1.0 mg/L for all endpoints.