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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 April 2018 to ****

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

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:

Samples were taken from the control and each loading rate WAF test group from the freshly prepared bulk test solutions at 0 and 24 hours from the old solutions as pooled replicates at 24 and 48 hours, and for quantitative analysis. Samples were stored frozen prior to analysis.

Vehicle:

no

Details on test solutions:

Due to the LOQ 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 were each separately added to the surface of test water to give the 1.0, 3.2, 10, 32, and 100 mg/L. loading rates WAFs, respectively. After the addition of the test item, the test water 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 column and hence it was considered justifiable to remove the dispersed material by filtering the WAFs through a glass wool plug. 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. The aqueous phase or WAF was removed by mid depth siphoning (the first 75 to 100 mL discarded) to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAFs.

Micropsopic observations of the WAFs were performed after filtering and showed no undissolved test item to be present.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water Flea
- Source: In-house laboratory culture
-Culture medium (dilution water): Elendt M7 medium
-Culture conditions: 18 to 22°C, 16 hours light and 8 hours darkness cycle with 20 minute dawn and dark transition periods
- Age: Less than 24 hours old
- Food: mixture of algal suspension (Desmodesmus subspicatus) and Tetramin flake food suspension
- Frequency: Daily for culture, the daphnids received no food during exposure

The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

20 - 21°C

pH:

7.7 - 8.1

Dissolved oxygen:

8.7 - 9.1 mg O2/L

Nominal and measured concentrations:

Based on the results of the rang-finding test the following nominal loading rates were employed in the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L.

Chemical analysis of the freshly prepared test preparations at 0 and 24 hours, and of the old or expired test preparations at 24 and 48 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed (determined to be 0.040 mg/L) were obtained.

This does not suggest that no test item was in solution, just that the marker constituent 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.

Details on test conditions:

TEST SYSTEM
Test solutions were prepared in the same manner as described for the range-finding test. Jars (150 mL) containing approximately 100 mL of test solution were used. Within 30 minutes of the test media being added to the test vessels, five daphnids were placed at random in each test and control vessel containing the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room maintaining the water temperature at 18 to 22°C with a maximum deviation of ±1°C and a photoperiod of 16 hours light (between 200 and 1200 lux) and 8 hours darkness with 20 minute dawn and dust transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.

The control group was maintained under identical conditions, but not exposed to the test item.

Semi-static test conditions were employed in the test in an effort to maintain dissolved test item concentrations. For the test media renewal at 24 hours, the test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24 hour old test media into the fresh test media. Concentrations at which 100% immobilization was observed after 24 hours exposure were not renewed.

OTHER TEST CONDITIONS
The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours and after the test media renewal at 24 hours represent those of the freshly prepared test preparations while the measurements taken prior to the test media renewal, and on termination of the test after 48 hours, represent those of the used or 24 hour old test preparations. The appearance of the test media was recorded daily.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

45 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(dissolved fraction) loading rate WAF

Basis for effect:

mortality

Key result

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(dissovled fraction) loading rate WAF

Basis for effect:

mortality

Key result

Duration:

48 h

Dose descriptor:

LOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(dissolved fraction) loading rate WAF

Basis for effect:

mortality

Details on results:

RANGE-FINDING TEST
No immobilization was observed at 1.0 mg/L loading rate WAF, however, immobilization was observed at 10 and 100 mg/L loading rate WAF.
A sub-lethal effect of exposure observed in the 10 mg/L loading rate was trapping at the surface of the test material.
Chemical analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be less than the preliminary LOQ of the analytical method employed, which was later confirmed to be 0.040 mg/L upon validation of the method. As such, it was considered appropriate to conduct the definitive test employing a semi-static regime to maintain as much dissolved test item in solution as possible.
Based on results of the range finding test, loading rates of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test.

INITIAL TEST
Initial tests were conducted at the following loading rates: 1.0, 3.2, 10, 32 and 100 mg/L.
The results of the first test showed an inconsistent immobilization pattern and the results of the second test showed more than 10% of the control group were immobilized.

The results from the initial tests were not used for reporting purposes.


DEFINITIVE TEST
Sub-lethal effects of exposure observed in the 10 and 32 mg/L loading rate WAF groups included pale daphnids and reduced mobility.

Results with reference substance (positive control):

A positive control using potassium dichromate as the reference item, was conducted at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L. Expsure conditions for the positive control were similar to those in the definitive test. The NOEC is based upon equal to or less than 10% immobilization at this concentration. The results from the positive control with potassium dichromate were within the normal range for this reference item.

Reported statistics and error estimates:

An estimate of the loading rate causing 50% immobilization in the treatment group (EL50) at 24 and 48 hours, as well as, the slope of the response curve and its standard error, were calculated by Probit analysis using linear Maximum-Likelihood regression. The Lowest Observed Effect Loading rate (LOEL) and the No Observed Effect Loading rate (NOEL) at 24 and 48 hours were calculated using the Step-down Cochran-Armitage Test Procedure. All results were calculated using the ToxRat Professional computer software package (TOXRAT).

Cumulative Immobilization Data and Observations in the Definitive Test

Nominal Loading Rate (mg/L)	24 Hours
	Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)						Observations
	Rep1	Rep2	Rep3	Rep4	Total	%	Rep1	Rep2	Rep3	Rep4
Control	0	0	0	0	0	0	5N	5 N	5 N	5 N
1.0	0	0	0	0	0	0	5 N	5 N	5 N	5 N
3.2	1	0	0	0	1	5	4 N	5 N	5 N	5 N
10	0	0	0	0	0	0	5 N	5 N	5 N	5 N
32	0	0	0	0	0	0	5 N	5 N	5 N	5 N
100	5	5	5	5	20	100	AI	AI	AI	AI
Nominal Loading Rate (mg/L)	48 Hours
	Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)						Observations
	Rep1	Rep2	Rep3	Rep4	Total	%	Rep1	Rep2	Rep3	Rep4
Control	1	0	0	0	1	5	4 N	5 N	5 N	5 N
1.0	1	0	0	0	1	5	4 N	5 N	5 N	5 N
3.2	1	0	0	0	1	5	4 N	5 N	5 N	5 N
10	0	0	0	0	0	0	5 P	5 P	5 N	5 N
32	2	0	0	0	2	10	2 N 1 R	5 P	5 P	5 P
100	5	5	5	5	20	100	AI	AI	AI	AI

Rep    = Replicate

N          = Normal

P           = Pale

R          = Reduced mobility

AI        = All daphnia immobilized

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Daphnia magna to the test item for 48 hours resulted in an EL50 value of 45 mg/L loading rate WAF. The No Observed Effect Loading Rate was 32 mg/L loading rate WAF.

Executive summary:

A study was conducted in accordance with OECD Chemicals Testing Guideline No. 202 and EU Test Method C.2, under GLP conditions, to assess the acute toxicity of the test item to the freshwater invertebrate, Daphnia magna. Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test and initial tests, 20 daphnids (4 replicates of 5 animals) were expsoed to nominal WAF loading rates of 1.0,3.2, 10, 32 and 100 mg/L for 48 hours at a temperature of 20°C to 21°C under semi-static test conditions. The number of immobilized daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours.

The test item concentrations in the test samples was determined by HPLC-MS using an external standard and quantitation was based on the peak of the major constituent detected.

Chemical analysis of the freshly prepared test preparations at 0 and 24 hours, and of the old or expired test preparations at 24 and 48 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed (determined to be 0.040 mg/L) were obtained.  This does not suggest that no test item was in solution, just that the marker constituent 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 Daphnia magna to the test item for 48 hours resulted in EL50 value of 45 mg/L loading rate WAF and the No Observed Effect Loading Rate (NOEL) was 32 mg/L loading rate WAF.

Description of key information

A study was conducted according to OECD Guideline 202 and EC Method C.2, under GLP, to assess the acute toxicity of the test item to the freshwater invertebrate,Daphnia magna. Exposure ofDaphnia magnato the test item for 48 hours resulted in EL50 value of 45 mg/L loading rate WAF and the No Observed Effect Loading Rate was 32 mg/L loading rate WAF.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

45 mg/L