Selenium disulphide

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:

15 January 2018 to 18 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

OECD Guideline for Testing of Chemicals, Section 2, No. 201: "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", adopted 28th July, 2011.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Commission Regulation (EC) No 761/2009 of 23 July 2009 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACh), Annex IV Part C, C.3 (published in the Official Journal of the European Union L 220 of 24 August 2009).

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Version / remarks:

EPA Ecological Effects Test Guidelines, OCSPP 850.5400, Algal Toxicity, Tiers I and II, EPA 712-C-96-164, April 1996.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

Analytical monitoring:

yes

Details on sampling:

The concentration of the Test Item was measured at the tested concentration level at the beginning and the end of the test.

Vehicle:

no

Details on test solutions:

Because the Test Item was very poorly soluble in water (see water solubility study performed in Seibersdorf Laboratories, Citoxlab study code: 17/003-910AN), a test solution was prepared using a saturated solution method according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23.
A saturated Test Item solution (nominal loading rate of 100 mg/L) was prepared by dispersing/dissolving the amount of Test Item into the test medium (OECD Medium) two days before the start of the experiment. This solution was shaken for approximately 24 hours at approximately 30°C (Day -2) and then was equilibrated for approximately 24 hours at approximately 20°C (Day -1). The non-dissolved test materials were removed by filtration through a fine (0.22 µm) filter to give the 100% saturated solution.
As only limit test was carried out, further dilution of this saturated solution was not performed. Test solution was distributed into test vessels prior to introduction of algae.

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum)
Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
Source: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, and University of Göttingen, GERMANY. Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of Citoxlab Hungary Ltd.
Justification of species: The species of Pseudokirchneriella subcapitata used, being a fast-growing species, is convenient for culturing and testing and is a recommended species by relevant guidelines.
Breeding conditions: Stock cultures are small algal colonies that are inoculated onto agar regularly. These are transferred to fresh agar medium at least once every two months and are maintained under standardised conditions according to the test guidelines.
The pre-culture is intended to give a quantity of algae suitable for the inoculation of test cultures. The pre-culture was prepared with the OECD algal growth medium, incubated under the same conditions as the test and used when still growing exponentially, normally after an incubation period of about three days. When the algal cultures contain deformed or abnormal cells, they were discarded.

Test type:

not specified

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

Post exposure observation period not specified in the study report.

Hardness:

Not specified

Test temperature:

The temperature was in the range of 22.7 – 22.8°C measured in the flask and 22.4 – 23.0°C between measured within the climate chamber.

pH:

The range of the pH was 7.23 – 8.61 during the experiment.

Dissolved oxygen:

Not specified

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

One test concentration at 100% saturated solution (from a Loading Rate of 100 mg/L) and one control group was tested in a Limit Test.

Details on test conditions:

Test conditions
Temperature
Culture temperature was checked at the beginning of the experiment and each day thereafter in a flask filled with water, in the climatic chamber. In addition, water temperature was continuously measured (with a min/max thermometer) within the climate chamber. The temperature was in the range of 22.7 – 22.8 °C measured in the flask and 22.4 – 23.0 °C between measured within the climate chamber.

pH
The pH was checked at the beginning and at the end of the test, in the control and each concentration. The pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 7.23 – 8.61 during the experiment.

Light Intensity
The algal culture flasks were continuously illuminated. The light intensity at the position occupied by algal culture flasks during the test was about 7813 lux (equivalent to ~105.6 μE/m2/s), which was ensured with fluorescent lamps (with a spectral range of 400-700 nm). The differences in light intensity between the test vessels did not exceed ± 15 % and therefore provided equal conditions for each test vessel.

Performance of the test
The exposure time was 72 hours. The test was started (0 hours) by inoculation of a biomass of approximately 10e+4 algal cells per mL test medium.
The test was performed with six replicates per test concentration and six replicates in the control group as well. Volumes of 100 mL test solution per replicate in 250 mL Erlenmeyer flasks were continuously shaken by a laboratory orbital shaker to keep algae in suspension. The flasks were covered with air-permeable stoppers.

Preliminary Range-Finding Test
A concentration range-finding test was conducted to determine the approximate toxicity of the Test Item so that appropriate test concentrations could be selected for use in the definitive test. Algal cells were exposed to each concentration of the Test Item plus a control, for 72 hours. The test was performed with two replicates per each test concentration and three replicates in the control group.
During the formulation procedure the stock solution was prepared and the test solutions were prepared by appropriate dilution of this stock solution.

Test Item concentrations in the Definitive Test
Because significantly toxic response was not observed during the preliminary concentration Range-Finding Test, only one test concentration at 100% saturated solution and one control group will be tested in a Limit Test.

Observations
The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber.
Microscopic observation of the algal cells in each concentration and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 other: % saturated solution (based on 100 mg/L loading rate)

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 other: % saturated solution (based on 100 mg/L loading rate)

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % saturated solution (based on 100 mg/L loading rate)

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

VALIDITY
The cell density in the control cultures increased by the factor of 70.00 within three days.
The mean coefficient of variation for section-by-section specific growth rates (days 0-1; 1-2; 2-3) in the control cultures was 10.21 %.
The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 0.70 %.
All validity criteria were met; therefore, the study can be considered as valid.

MORPHOLOGICAL DEVIATIONS OF THE ALGAL CELLS
There were no morphological deviations of the algal cell during the study in any of the test groups.

AVERAGE SPECIFIC GROWTH RATES
The results of the statistical evaluation (based on 2 Sample t-Test; α=0.05) show that the 0-72 h average specific growth rate was not statistically significantly different from the untreated control value, accordingly the No Observed Effect Concentration (NOEC) was determined as 100 % saturated solution (from a nominal loading rate of 100 mg/L).

AREAS UNDER THE GROWTH CURVES
The results of the statistical evaluation (based on 2 Sample t-Test; α=0.05) show that the 0-72 h areas were not statistically significantly different from the untreated control value, accordingly the No Observed Effect Concentration (NOEC) was determined as 100 % saturated solution (from a nominal loading rate of 100 mg/L ).

YIELD
The results of the statistical evaluation (based on 2 Sample t-Test; α=0.05) show that the 0-72 h yield was not statistically significantly different from the untreated control value; accordingly the No Observed Effect Concentration (NOEC) was determined as 100% saturated solution (from a nominal loading rate of 100 mg/L ).

Results with reference substance (positive control):

The 72h ErC 50: 0.88 mg/L, (95 % confidence limits: 0.81 – 0.96 mg/L)
The 72h EbC 50: 0.63 mg/L, (95 % confidence limits: 0.58 – 0.69 mg/L)
The 72h EyC 50: 0.53 mg/L, (95 % confidence limits: 0.49 – 0.58 mg/L)

Reported statistics and error estimates:

The section-by-section specific growth rates in the control cultures were assessed (calculated as the specific growth rates for each day during the course of the test (days 0-1, 1-2 and 2-3) and to demonstrate exponential growth for the entire study period.
The inhibition of alga growth was determined from the biomass (area under the growth curves, A), the average specific growth rate (r) and from the yield (y). Mean values and standard deviations were calculated for each concentration at the start, and at the end of the test using Excel for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399).
The ErC50, EbC50 and EyC50 values of the Test Item were determined from the raw data.
Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using 2 Sample t-Test (α = 0.05) by TOXSTAT software.
For the determination of the LOEC and NOEC, the calculated mean biomass, growth rates and yield at the test concentrations were tested on significant differences to the control values by 2 Sample t-Test (α = 0.05) by TOXSTAT software.

Result of the Preliminary Range-Finding Test

Nominal concentration [% saturated solution]	Untreated control	0.1	1	10	100
Average of cell number at 72 hours(x 10^4cell/mL)	69.0	71.0	68.5	67.5	66.5

 

Growth Rates (μ) and Percentage Inhibition of μ during the Test Period

Test group	Growth rate μ and % inhibition of μ
	0-24 h		0-48 h		0-72 h
	μ	%	μ	%	μ	%
Control	0.0573	0.0	0.0603	0.0	0.0590	0.0
100 % saturated solution (nominal)	0.0553^+	3.5	0.0600	0.5	0.0588	0.3

⁺: at this value the rounding of the EXCEL and TOXSTAT software was different. The table contains the values calculated with EXCEL.

 

Area under the Growth Curves (A) and Percentage Inhibition of A during the Test Period

Test group	Areas under the Growth Curves (A) and & inhibition of A
	0-24 h		0-48 h		0-72 h
	A	%	A	%	A	%
Control	36.0	0.0	278.0	0.0	1312.0	0.0
100 % saturated solution (nominal)	34.0	5.6	270.0	2.9	1288.0	1.8

 

Yield (Y) and Percentage Inhibition of Y during the Test Period

Test group	Yield (Y) and % inhibition of Y (0 – 72 h)
	Y	%
Control	69.0	0.0
100% saturated solution (nominal)	68.0	1.4

 

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of this study, the ErC50, EbC50 and EyC50 values of the Test Item were determined directly from the raw data.

The 72h EbC50 value: > 100 % saturated solution (Based on a 100 mg/L loading rate; the solution is considered to be saturated.)
The 72h ErC50 value: > 100 % saturated solution (Based on a 100 mg/L loading rate; the solution is considered to be saturated.)
The 72h EyC50 value: > 100 % saturated solution (Based on a 100 mg/L loading rate; the solution is considered to be saturated.)
The 72h NOEC: 100 % saturated solution (Based on a 100 mg/L loading rate; the solution is considered to be saturated.)
The 72h LOEC: > 100 % saturated solution (Based on a 100 mg/L loading rate; the solution is considered to be saturated.)

Based on the results of this study, the Test Item had no toxic effect at saturation; the EC50 results and the LOEC are higher than the solubility level of the Test Item in the test medium. The selenium content of the measured vessels were <0.0046 mg/L in each case.

Executive summary:

The effect of the Test Item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata, over an exposure period of 72 hours.

Because significantly toxic response was not observed during the preliminary concentration Range-Finding Test, only one test concentration at 100% saturated solution (from a Loading Rate of 100 mg/L) and one control group was tested in a Limit Test.

 

Since the test item is very poorly soluble in water and due to that there was no toxic effect at the solubility level of the test item in the test medium during the Preliminary Range-Finding Test the experiment will be carried out without analytical measurements in agreement with the Sponsor.

The test design included 6 replicates at test concentration and 6 replicates for the untreated control.

Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using 2 Sample t-Test (a= 0.05) by TOXSTAT software.

Under the conditions of this study, the ErC50, EbC50 and EyC50 values of the Test Item were determined directly from the raw data.

The 72h EbC50 value: > 100 % saturated solution (Based on a 100 mg/L loading rate; the solution is considered to be saturated.)

The 72h ErC50 value: > 100 % saturated solution (Based on a 100 mg/L loading rate; the solution is considered to be saturated.)

The 72h EyC50 value: > 100 % saturated solution (Based on a 100 mg/L loading rate; the solution is considered to be saturated.)

The 72h NOEC: 100 % saturated solution (Based on a 100 mg/L loading rate; the solution is considered to be saturated.)

The 72h LOEC: > 100 % saturated solution (Based on a 100 mg/L loading rate; the solution is considered to be saturated.)

 

Based on the results of this study, the Test Item had no toxic effect at saturation; the EC50 results and the LOEC are higher than the solubility level of the Test Item in the test medium. The selenium content of the measured vessels were <0.0046 mg/L in each case.

Description of key information

The 72h ErC50 value: > 100 % saturated solution (Based on a 100 mg/L loading rate; the solution is considered to be saturated.)

The 72h NOEC: 100 % saturated solution (Based on a 100 mg/L loading rate; the solution is considered to be saturated.)

The 72h LOEC: > 100 % saturated solution (Based on a 100 mg/L loading rate; the solution is considered to be saturated.)

Additional information

The effect of the Test Item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata, over an exposure period of 72 hours.

Because significantly toxic response was not observed during the preliminary concentration Range-Finding Test, only one test concentration at 100% saturated solution (from a Loading Rate of 100 mg/L) and one control group was tested in a Limit Test.

 

Since the test item is very poorly soluble in water and due to that there was no toxic effect at the solubility level of the test item in the test medium during the Preliminary Range-Finding Test the experiment will be carried out without analytical measurements in agreement with the Sponsor.

The test design included 6 replicates at test concentration and 6 replicates for the untreated control.

Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using 2 Sample t-Test (a= 0.05) by TOXSTAT software.

Under the conditions of this study, the ErC50, EbC50 and EyC50 values of the Test Item were determined directly from the raw data.

The 72h EbC50 value: > 100 % saturated solution (Based on a 100 mg/L loading rate; the solution is considered to be saturated.)

The 72h ErC50 value: > 100 % saturated solution (Based on a 100 mg/L loading rate; the solution is considered to be saturated.)

The 72h EyC50 value: > 100 % saturated solution (Based on a 100 mg/L loading rate; the solution is considered to be saturated.)

The 72h NOEC: 100 % saturated solution (Based on a 100 mg/L loading rate; the solution is considered to be saturated.)

The 72h LOEC: > 100 % saturated solution (Based on a 100 mg/L loading rate; the solution is considered to be saturated.)

 

Based on the results of this study, the Test Item had no toxic effect at saturation; the EC50 results and the LOEC are higher than the solubility level of the Test Item in the test medium. The selenium content of the measured vessels were <0.0046 mg/L in each case.