Polysulfides, di-tert-Bu

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:

2012-09-06 to 2012-11-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

30/11/12

Test material

Test material information

Constituent 1

Details on test material:

- Description: amber colored liquid- Purity: not applicable - complex mixture- Expiry / Retest Date: 25 August 2013- Storage Conditions: room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Samples were taken from the control and the 100 mg/L loading rate WAF test group at 0 and 72 hours for quantitative analysis- Experimental samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter Multisizer Particle Counter- All samples were stored at approximately -20°C prior analysis

Test solutions

Vehicle:

no

Details on test solutions:

- An amount of 200mg of test tiem was added to the surface of 2 liters of culture medium- Culture medium of 100 mg/L was stirred by magnetic stirrer during 23 hours and then allowed to stand during 1 hour- a wide bore glass tube, covered at one end with Nexcofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inseted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 100 mg/L loading rate WAF. - An aliquot (1 liter) of the WAF was inoculated with algal suspension (7.6 ml) to give the required test concentration of 100 mg/L loading rate WAF.- The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours

Test organisms

Test organisms (species):

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

Details on test organisms:

Liquidi cultures of Pseudokirchnerella subcapitata strain CCAP 278/4 were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.Cultures were mantained under constant aeration and illumination at ca. 21°C

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

24 +/- 1°C

pH:

pH values of the control increase from 7.8 at 0 hours to 8.3 at 72 hourspH values of 100 mg/L increase from 7.7 at 0 hours to 8.2 at 72 hours

Dissolved oxygen:

Not specified

Salinity:

Not applicable

Nominal and measured concentrations:

Analysis of the 100 mg/L loading rate WAF at 0 hours showed a measured test concentration of 1.0 mg/L was obtained. A declin in measured test concentration was observed at 72 hours to 0.012 mg/L.

Details on test conditions:

TEST SYSTEM- Test vessel: 250 mL conical flasks- Type (delete if not applicable): closed- Material, size, headspace, fill volume: 100 mL of test preparation- Initial cells density:5.03 x 10^3 cells/mL- Control end cells density: 7.22 x 10^5 cells/mL- No. of vessels per concentration (replicates): 6- No. of vessels per control (replicates): 6GROWTH MEDIUM- Standard medium used: yesOTHER TEST CONDITIONS- Sterile test conditions: no- Adjustment of pH: no data- Photoperiod: constant illumination- Light intensity and quality: 7000 luxEFFECT PARAMETERS MEASURED (with observation intervals if applicable) :- Determination of cell concentrations: Counting chamberTEST CONCENTRATIONS- Range finding study- Test concentrations: 10, 100 mg/L- Results used to determine the conditions for the definitive study: no significant inhibition of growth at 10 mg/L loading rate WAF.

Reference substance (positive control):

yes

Remarks:

A positive control used potassium dichromate as the reference item.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of growth rate- loading rate WAF

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of yield - loading rate WAF

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: loading rate WAF

Details on results:

Pre-study work indicted taht there was no significant increase in the amount of total organic carbon by extending the preparation period for longer than 24 hours. The results of range-finding test show no effect on growth at 10 and 100 mg/L loading rate WAF. Based on this 6 replicates was selected for the definitive limit test to confirm that no effect on growth are observed. A decline in these measured test concentration was observed at 72 hours to less than the limit of quantification.The cell concentration of the control cultures increased by a factor of 144 after 72 hours. The mean coefficient of variation for section by section specific growth rate for the control cultures was 20%. The coefficient of variation for averge specific growth rate for the control cultures over the test period (0-72 h) was 1%- Inhibition of growth rate: Statistical analysis of the growth rate data was carried out for the control and 100 mg/L loding rate WAF test group using a Student's t-test incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparison procedure for comparing several treatments with a control. There were no statistically significant differences (P>0.05), between the control and 100 mg/L loading rate WAf test group and therefore the "No Observed Effect Loading Rate" based on growth rate was 100 mg/L loading rate WAF.- Inhibition of yield: There were no statistically significant differences between the control and 100 mg/L loading rate WAF (P>0.005). Therefore the "No Observed Effect Loading Rate" based on yield was 100 mg/L loading rate WAF.Observation on the test media were carried out during mixing and testing on the WAF. At the start of stirring the 100 mg/L loading rate WAf was observed to have formed a clear colorless media column with test item at the surface and dispersed troughout. After stirring and following a 1 hour settlement period the WAF was observed to have formed a clear colorless media column with globules of test item at the surface and settled on the base of mixing vessel. Microcopic examination of the WAF showed there to be no globules or-dipersions of the test item present. After 72 hours test period all control and test cultures were observed to be pale green dispersions.Analysis of the test loading rates WAF at 0 hours shows a mesured concentration of 1.0 mg/L and then a decline at 72 hours to 0.012 mg/L. Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Results with reference substance (positive control):

DurationEndpointEffect conc.Nominal/MeasuredConc. based onBasis for effectRemarks (e.g. 95% CL)72 hEC501.1 mg/Lnominaltest mat.biomass95% confidence limits 1.0 - 1.3 mg/L72 hEC500.7 mg/Lnominaltest mat.growth rateNot possible to calculate 95% confidence limits for EC5072 hNOEC0.5 mg/Lnominaltest mat.growth rate72 hNOEC0.25 nominaltest mat.biomass72 hLOEC1 mg/Lnominaltest mat.growth rate72 hLOEC0.5 mg/Lnominaltest mat.biomassA positive control used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L. Exposure conditions and data evaluation for the positive control were similar to those in the definitive test. The results from the positive control with potasium dichromate were within the normal ranges for this reference item.

Any other information on results incl. tables

Cell Densities an percentag inhibition of Growth from the range-finding test

Nominal Loading Rate (mg/L)		Cell Densities µ (cells per mL)		Inhibition Values (%)
		0 hours	72 Hours	Growth Rate	Yield
Control	R1	5.81E+03	5.95E+05
	R2	5.10E+03	7.42E+05
	Mean	5.46E+03	6.69E+05
10	R1	5.30E+03	6.95E+05
	R2	5.39E+03	9.01E+05
	Mean	5.34E+03	7.98E+05
100	R1	5.34E+03	5.82E+05
	R2	5.01E+03	7.06E+05	4	19
	Mean	5.18E+03	6.44E+05	0	4

Cell density an pH values in the definitive test

Nominal Loading Rate (mg/L)		Ph	Cell Densities µ (cells per mL)				Ph
		7.8	0 hours	24 Hours	48 Hours	72 Hours	8.3
Control	R1		4.77E+03	2.01E+04	1.15E+05	6.71E+05
	R2		4.98E+03	2.15E+04	1.30E+05	7.64E+05
	R3		5.46E+03	1.78E+04	1.38E+05	7.66E+05
	R4		5.02E+03	1.62E+04	1.2E+05	6.85E+05
	R5		5.04E+03	1.95E+04	1.34E+05	8.01E+05
	R6		4.91E+03	1.64E+04	1.33E+05	6.46E+05
	Mean		5.03E+03	1.86E+04	1.28E+05	7.22E+05
100	R1	7.7	4.74E+03	1.61E+04	1.11E+05	6.92E+05	8.2
	R2		4.10E+03	1.87E+04	9.22E+04	6.83E+05
	R3		5.02E+O3	1.83E+04	1.02E+05	6.49E+05
	R4		4.99E+03	1.85E+04	1.06E+05	7.11E+05
	R5		5.01E+03	1.83E+04	9.57E+04	6.52E+05
	R6		5.07E+03	1.56E+04	1.10E+05	5.48E+05
	Mean		4.82E+03	1.76E+04	1.03E+05	6.56E+05

Daily specific growth rate for the control cultures in the definitive test

		Daily specific growth rate (cells/mL/hour)
		Day 0-1	Day 1 - 2	Day 2 - 3
Control	R1	0.058	0.073	0.073
	R2	0.061	0.075	0.074
	R3	0.053	0.085	0.071
	R4	0.049	0.083	0.073
	R5	0.057	0.080	0.075
	R6	0.050	0.087	0.066
	Mean	0.055	0.081	0.072

Inhibition of growth rate and yield in the definitive test

Nominal Loading Rate (mg/L)		Growth rate (cells per mL per hour)		Yield (cells/mL)
		0 - 72hours	% inhibition	0 - 72 h	% inhibition
Control	R1	0.068		6.66E+05
	R2	0.070		7.59E+05
	R3	0.070		7.61E+05
	R4	0.068		6.80E+05
	R5	0.071		7.96E+05
	R6	0.068		6.42E+05
	Mean	0.069		7.17E+05
	SD	0.001		6.26E+04
100	R1	0.068	1	6.87E+05
	R2	0.068	1	6.78E+05
	R3	0.068	1	6.44E+05
	R4	0.069	0	7.06E+05
	R5	0.068	1	6.47E+05
	R6	0.065	6	5.43E+05
	Mean	0.068	2	6.51E+05	9
	SD	0.001		5.80E+04

Vrtex deph measurements at the start and end of the mixing period

	Nominal loading rate (mg/L)
	Control		100
	*	+	*	+
Height of media column (cm)	10.0	12.5	10.0	12.5
Deph of Vortex (cm)	ca. 0.2	ca. 0.2	ca. 0.2	ca. 0.2
Observation of Vortex	Dimple present	Dimple present	Dimple present	Dimple present

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Pseudokirchnerella subcapitata to the test material gave an EL*50 (72 h) greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

Executive summary:

Methods:A study was performed to assess the effect of the test material on the growth of the green alga Pseudokirchnerella subcapitata. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 201, "Alga, Growth Inhibition Test" referenced as Method C.3 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Procedure:Following a preliminary range-finding study, Pseudokirchnerella subcapitata was exposed to Water-Accommodated Fraction (WAF) of the test material at a single nominal loading rate of 100 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 +/-1°C. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group using a Coulter@ Multisizer Particle Counter.

Results: Exposure of Pseudokirchnerella subcapitata to the test material gave an EL*50 (72 h) values greater than 100 mg/L was obtained. loading rate WAF. The No Observed Effect Loading Rate was 100mg/L loading rate WAF.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

Analysis of the 100 mg/L loading rate WAF at 0 hours showed a measured test concentration of 10 mg/L was obtained. A decline in measured test concentration was observed at 72 hours to 0.012 mg/L.

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.