Reaction products of 2,5-dimercapto-1,3,4-thiadiazole, sodium salt, with 1-octanethiol and hydrogen peroxide

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:

10 November 2017 to ****

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Physical state/Appearance: Clear, yellow liquid
Purity: >99% (UVCB)

Analytical monitoring:

yes

Details on sampling:

The concentrations and stability of the test solutions were assessed by chemical analysis at 0 and 72 hours.

Samples (50 mL) were taken from the control and each test group from the bulk test solutions at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at each occasion and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

Due to the low aqueous solubility and the complex nature of the test item, the test medium was prepared as Water Accommodating Fraction (WAF) based on the methods established in a preliminary mixing trial.

Nominal amounts of test item (50, 90, 160 and 500 mg) were each separately added to the surface of 5 liters of culture medium to achieve the 10, 18, 32 and 100 mg/L loading rates respectively. In error, a nominal amount of test item (56 mg) was weighed out and added to the surface of 5 liters of culture media; this gave a nominal loading rate of 11 mg/L. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. Microscopic observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water columns of the 32, 11 and 100 mg/L loading rates and hence it was considered justifiable to remove this material by filtering the WAFs through a glass wool plug (2 to 4 cm in length). A wide bore glass tube, covered at one end with Nescofilm 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 inserted into the glass tube and pushed through the Nescofilm seal, a glass wool plug was inserted into the opposite end of the tubing and the 32, 11 and 100 mg/L loading rate WAFs removed by mid depth siphoning (the first 75 to 100 mL discarded). Microscopic inspection of the 10 and 18 mg/L WAFs showed no micro dispersions or undissolved test item to be present hence it was considered unnecessary to filter these through a glass wool plug. Microscopic observations of the 32, 11 and 100 mg/L loading rate WAFs was performed after filtering and showed no micro dispersions of test item to be present.

An aliquot (1 liter) of each of the loading rate WAFs was separately inoculated with algal suspension (5.1 mL) to give the test concentrations of 10, 18, 32, 11 and 100 mg/L loading rate WAF.

The concentration and stability of the test item in the test preparations were assessed by chemical analysis at 0 and 72 hours.

Test organisms (species):

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

Details on test organisms:

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant agitation by orbital shaker (100 to 150 rpm) and constant illumination at 24 +/- 1 °C.

Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 to 150 rpm) and constant illumination at 24 +/- 1 °C until the algal cell density was approximately 10^4 to 10^5 cells/mL.

Test type:

static

Water media type:

other: algal culture medium

Total exposure duration:

72 h

Test temperature:

24 +/- 1 °C

pH:

7.4 - 8.6

Nominal and measured concentrations:

Nominal concerntrations were 0 (control),10, 18, 32, 11* and 100 mg/L loading rate WAF.

During the definitive test, the 56 mg/L was dosed incorrectly resulting in a nominal loading rate of 11 mg/L. Given that no toxicity was observed at any of the loading rates tested, this error was considered not to have had an impact on the outcome or integrity of the test.

Details on test conditions:

As in the range-finding tests 250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and three flasks each containing 100 mL were used for each treatment group.

The control group was maintained concurrently under identical conditions, but was not exposed to the test item.
Pre culture conditions gave an algal suspension in log phase growth characterized by a cell density of 9.83 x 10^5 cells per mL. Inoculation of 1L of test medium with 5.1 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10^3 cells per mL and had no significant dilution effect on the final test concentration.

The flasks were plugged with polyurethane foam bungs, incubated at 24 +/- 1 °C under continuous illumination provided by warm white lighting, and constantly shaken at approximately 150 rpm for 72 hours.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(loading rate WAF)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(loading rate WAF)

Basis for effect:

growth rate

Details on results:

There were no statistically significant differences (P>=0.05), between the control and any of the loading rate WAF test groups and therefore the (NOEL) based on growth rate was 100 mg/L loading rate WAF.

There were no statistically significant differences between the control and any of the loading rate WAF test groups (P>/=0.05); the NOEL based on yield was therefore determined to be 100 mg/L loading rate WAF.

There were no statistically significant differences between the control and all of the loading rate WAF test groups (P>/=0.05); the NOEL based on biomass integral was therefore determined to be 100 mg/L loading rate WAF.

The cell concentration of the control cultures increased by a factor of 251 after 72 hours. The mean coefficient of variation for section by section specific growth rate for the control cultures was 7% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%. The coefficient of variation for average specific growth rate for the control cultures over the test period (0 to 72 hour) was 1% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-hour test period all control and test cultures were observed to be green dispersions.

Results with reference substance (positive control):

A positive control, using potassium dichromate as the reference item, was conducted 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. Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 to 72 hour): 1.6 mg/L; 95% confidence limits 1.4 to 1.8 mg/L
EyC50 (0 to 72 hour): 0.77 mg/L; 95% confidence limits 0.68 to 0.87 mg/L

NOErC (based on growth rate): 0.25 mg/L
NOEyC (based on yield): 0.25 mg/L

LOErC (based on growth rate): 0.50 mg/L
LOEyC (based on yield): 0.50 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Loading Rate (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)		Biomass Integral
		0 to 72 Hour	% Inhibition	0 to 72 Hour	% Inhibition*	0 - 72 h	% Inhibition
Control	R1	0.077	-	1.28E+06	-	2.16E+07	-
	R2	0.076		1.21E+06		2.06E+07
	R3	0.077		1.26E+06		2.11E+07
	R4	0.075		1.11E+06		1.96E+07
	R5	0.078		1.34E+06		2.26E+07
	R6	0.077		1.30E+06		2.18E+07
	Mean	0.077		1.25E+06		2.12E+07
	SD	0.001		8.13E+04		1.06E+06
10	R1	0.076	1	1.15E+06		2.00E+07	5
	R2	0.075	3	1.12E+06		1.96E+07	7
	R3	0.080	[4]	1.55E+06		2.57E+07	[21]
	Mean	0.077	0	1.27E+06	[2]	2.18E+07	[3]
	SD	0.003		2.38E+05		3.39E+06
18	R1	0.078	[1]	1.38E+06		2.33E+07	[10]
	R2	0.078	[1]	1.41E+06		2.38E+07	[12]
	R3	0.078	[1]	1.40E+06		2.38E+07	[12]
	Mean	0.078	[1]	1.40E+06	[12]	2.36E+07	[11]
	SD	0.000		1.36E+04		2.92E+05
32	R1	0.077	0	1.26E+06		2.14E+07	[1]
	R2	0.075	3	1.09E+06		1.87E+07	12
	R3	0.079	[3]	1.49E+06		2.53E+07	[19]
	Mean	0.077	0	1.28E+06	[2]	2.18E+07	[3]
	SD	0.002		2.00E+05		3.32E+06
11	R1	0.075	3	1.12E+06		1.94E+07	8
	R2	0.073	5	9.70E+05		1.72E+07	19
	R3	0.080	[4]	1.60E+06		2.69E+07	[27]
	Mean	0.076	1	1.23E+06	1	2.12E+07	0
	SD	0.004		3.31E+05		5.09E+06
100	R1	0.079	[3]	1.43E+06		2.41E+07	[13]
	R2	0.077	0	1.29E+06		2.19E+07	[3]
	R3	0.078	[1]	1.33E+06		2.28E+07	[8]
	Mean	0.078	[1]	1.35E+06	[8]	2.29E+07	[8]
	SD	0.001		7.24E+04		1.08E+06

*In accordance with the OECD test guideline,only the mean value for yield for each test concentration is calculated

R   = Replicate

-    = Not applicable

SD   =Standarddeviation

[ ] = Increase in growth compared to controls

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Pseudokirchneriella subcapitata to the test item for 72 hours resulted in an EL50 of >100 mg/L loading rate WAF. The No Observed Effect Loading rate (NOEL) was therefore detremined to be 100 mg/L loading rate WAF.

Executive summary:

A study was conducted according to OECD Guideline 201 and EC Method C.3, under GLP conditions, to assess the effects of the test item on the growth of the freshwater green alga, Pseudokirchneriella subcapitata.  

 

Due to the low aqueous solubility and complex nature of the test item, the test solutions were prepared as Water Accommodated Fractions (WAF).

 

Following a preliminary range finding tests, Pseudokirchneriella subcapitata was exposed to Water Accommodated Fractions (WAFs) of the test item at nominal loading rates of 10, 18, 32, and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 ˚C. Samples of the test solutions were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

 

The test item concentration in the test samples was determined by high performance liquid chromatography with mass spectrometry (HPLC-MS) using an external standard; quantitation was based on the major constituent detected. Analysis of the 100 mg/L loading rate at 0 and 72 hours showed that measured concentrations of less than the Limit of Quantification (LOQ) of the analytical method employed (determined to be 0.021 mg/L) were obtained. This does not suggest that no test item was in solution, just that the marker constituents used for quantitation was present in the dissolved fraction at a concentration of less than the LOQ of the method employed. Given that the toxicity cannot be attributed to a single component or a specific mixture of components, the results were based on nominal loading rates.

 

Exposure of Pseudokirchneriella subcapitata to the test item for 72 hours resulted in an ErL50 of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate (NOEL) was 100 mg/L loading rate WAF.

Description of key information

A study was conducted according to OECD Guideline 201 and EC Method C.3, under GLP conditions, to assess the effects of the test item on the growth of the freshwater green alga, Pseudokirchneriella subcapitata. Exposure of Pseudokirchneriella subcapitata to the test item for 72 hours resulted in an EL50 of >100 mg/L loading rate WAF, and a NOEL of 100 mg/L loading rate WAF, based on growth rate.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L